Polyvalent conjugate vaccine for cancer

ABSTRACT

This invention provides a polyvalent vaccine comprising at least two conjugated antigens selected from a group containing glycolipid antigen, polysaccharide antigen, mucin antigen, glycosylated mucin antigen and an appropriate adjuvant. This invention also provides a multivalent vaccine comprising at least two of the following: glycosylated MUC-1-32mer, Globo H, GM2, Le y , Tn(c), sTN(c), and TF(c). This invention provides the vaccine above, wherein the adjuvant is saponin-based adjuvant. This invention provides a method for inducing immune response in a subject comprising administering an effective amount of the vaccine above to the subject. Finally, this invention provides a method for treating cancer in a subject comprising administering an appropriate amount of the vaccine above to the subject.

This application disclosed herein is a continuation of U.S. Ser. No. 10/752,945, filed Jan. 6, 2004 now U.S. Pat. No. 7,479,266, which is a continuation-in-part of International Application No. PCT/US02/21348, filed Jul. 5, 2002, which claims priority of U.S. Ser. 60/303,494, filed on Jul. 6, 2001 and U.S. Ser. No. 60/347,231, filed on Jan. 10, 2002, the contents of which are hereby incorporated by reference into this application.

Throughout this application, various references are referred to. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

The invention disclosed herein was made with United States government support under NIH Grant Nos. CA33049 and CA52477 from the United States Department of Health and Human Services. Accordingly, the United States Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Tumor-specific antigens have been identified and pursued as targets for vaccines. Previous work from the inventors' has shown that monovalent vaccines utilizing the tumor antigens Globo H, Lewis^(y), GM2, glycosylated MUC-1, Tn(c), sTn(c), or TF(c) conjugated to KLH to be safe with local erythema and edema but minimal systemic toxicities. As a result of vaccination with these monovalent vaccines, most patients generated specific high titer IgM or IgG antibodies against the respective antigen-KLH conjugates. The present invention provides a polyvalent vaccine wherein the components of the monovalent vaccines are combined and administered with an adjuvant as treatment for cancer.

SUMMARY OF THE INVENTION

The invention disclosed herein provides a polyvalent vaccine comprising at least two conjugated antigens selected from a group containing glycolipid antigen, polysaccharide antigen, mucin antigen, glycosylated mucin antigen and an appropriate adjuvant. This invention also provides the multivalent vaccine, comprising glycosylated MUC-1-32mer, Globo H, GM2, Le^(y), Tn(c), and TF(c). This vaccine may comprise glycosylated MUC-1-G5, Globo H, GM2, Le^(y), Tn(c), sTN(c), and TF(c). This invention provides the vaccine above, wherein the adjuvant is saponin-based adjuvant.

This invention also provides a method for inducing immune response in a subject comprising administering an effective amount of the vaccine above to the subject. Finally, this invention provides a method for treating cancer in a subject comprising administering an appropriate amount of the vaccine above to the subject.

DETAILED DESCRIPTION OF THE INVENTION

The invention disclosed herein provides a polyvalent vaccine comprising at least two conjugated antigens selected from a group containing glycolipid antigen, polysaccharide antigen, mucin antigen, glycosylated mucin antigen and an appropriate adjuvant.

The glycolipid includes but is not limited to Globo H, a Lewis antigen and a ganglioside. The Lewis antigen includes but is not limited to Le^(y) and sialyl Le^(a). The ganglioside includes fucosylated GM1, GM2, GD2, or GD3. In another embodiment, the mucin is a MUC peptide. In a further embodiment, the MUC peptide is MUC-1, MUC-2 or MUC-16. The polysaccharide antigen includes but is not limited to Tn(c), sTn(c), TF(c), and polysialic acid.

This invention provides a bivalent, trivalent, tetravalent, pentavalent, hexavalent, and heptavalent vaccine. The vaccine comprises at least two conjugated antigens selected from a group containing glycolipid antigen, polysaccharide antigen, mucin antigen, glycosylated mucin antigen and an appropriate adjuvant.

In an embodiment, the hexavalent vaccine comprises glycosylated MUC-1-32mer, Globo H, GM2, Le^(y), Tn(c), and TF(c). In a further embodiment, the range of MUC-1-32mer is from about 0.1 to 30 ug. In yet another embodiment, the range of Globo H is from about 0.1 to 10 ug. In still a further embodiment, the range of GM2 is from about 0.1 to 10 ug. In an additional embodiment, the range of Le^(y) is from about 0.1 to 60 ug. In a further embodiment, the range of Tn(c) is from about 0.1 to 10 ug. In an additional embodiment, the range of TF(c) is from about 0.1 to 30 ug.

In a separate embodiment, the adjuvant is saponin based. The adjuvant includes QS21 and GPI-0100. In an embodiment, the range of QS21 is from about 25 to about 200 ug. In another embodiment, QS21 is about 100 ug. In a separate embodiment, the adjuvant is GPI-0100 with a range from about 1 to 25 mg. In an embodiment, GPI-0100 is about 10 mg.

This invention provides a heptavalent vaccine comprising at least two conjugated antigens selected from a group containing glycolipid antigen, polysaccharide antigen, mucin antigen, and glycosylated mucin antigen and an appropriate adjuvant. In an embodiment, the vaccine comprises glycosylated MUC-1-G5, Globo H, GM2, Le^(y), Tn(c), sTN(c), and TF(c). In another embodiment, the range of MUC-1-G5 is from about 0.1 to 30 ug. In a further embodiment, the range of Globo H is from about 0.1 to 100 ug. In another embodiment, the range of GM2 is from about 0.1 to 10 ug. In still another embodiment, the range of Le^(y) is from about 0.1 to 60 ug. In an embodiment, the range of Tn(c) is from about 0.1 to 100 ug. In a further embodiment, the range of sTn(c) is from about 0.1 to 100 ug. In yet another embodiment, the range of TF(c) is from about 0.1 to 30 ug.

This invention provides the vaccine above, wherein the adjuvant is saponin-based adjuvant. These saponin-based adjuvants include but are not limited to QS21 and GPI-0100.

In an embodiment, the range of QS21 is from about 25 to 200 ug. In another embodiment, the QS21 is about 10 ug. In a separate embodiment, the adjuvant is GPI-0100 and the range is from about 1 to 25 mg. In a preferred embodiment, GPI-0100 is about 10 mg.

This invention provides a polyvalent vaccine comprising a conjugated glycosylated antigen, a conjugated ganglioside antigen and an appropriate adjuvant, wherein the antigens are conjugated to a carrier. In an embodiment, the carrier is Keyhole Limpet Hemocyanin (KLH).

This invention provides the polyvalent vaccine above comprising at least two conjugated antigens selected from a group containing glycolipid antigen, polysaccharide antigen, mucin antigen, and glycosylated mucin antigen and an appropriate adjuvant for cancer. In an embodiment, the cancer is prostate, breast or ovarian cancer.

This invention also provides a method for inducing immune response in a subject comprising administering an effective amount of the above vaccine to the subject.

Furthermore, this invention provides a method for treating cancer in a subject comprising administering an appropriate amount of the above vaccine to the subject.

This invention also provides a composition comprising the above vaccine and a carrier.

This invention also provides a pharmaceutical composition comprising the above vaccine and a pharmaceutically acceptable carrier.

In addition, the invention provides a vaccine for small cell lung cancer comprising at least two conjugated antigens selected from the group containing Globo H, fucosylated GM1, GM2, GD2, GD3, sialyl Le^(a) and polysialic acid. This invention also provides a method for inducing immune response in a subject bearing small cell lung cancer comprising administering an effective amount of the above vaccine to the subject. This invention furthermore provides a method for treating a subject bearing small cell lung cancer comprising administering an effective amount of the above vaccine to the subject.

In addition, this invention provides the above vaccine, further comprising an antigen selected from a group containing CA125, or a portion thereof, KSA peptide or protein, and PSMA, or a portion thereof.

This invention includes the above vaccines which further comprise other antigens which can induce antibody and/or immune response. As illustrated throughout the specification, the antigen used may be modified to increase its immunogenicity. Said antigens include but are not limited to CA125, or a portion thereof, KSA peptide or protein, and PSMA, or a portion thereof. As can be easily appreciated by the ordinary skilled artisan, only a portion of the antigen may be required for induction of immune response from a subject.

As stated herein, subjects are organisms which have immune response. The subject includes but is not limited to humans. Said subject could be domestic animals, such as dogs and cats.

This invention further provides the above compositions and a pharmaceutically acceptable carrier, thereby forming pharmaceutical compositions.

This invention also provides a pharmaceutical composition comprising a combination as described above and a pharmaceutically acceptable carrier. For the purposes of this invention, “pharmaceutically acceptable carriers” means any of the standard pharmaceutical carriers. Examples of suitable carriers are well known in the art and may include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution and various wetting agents. Other carriers may include additives used in tablets, granules and capsules, etc. Typically such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gum, glycols or other known excipients. Such carriers may also include flavor and color additives or other ingredients. Compositions comprising such carriers are formulated by well-known conventional methods.

The invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative, and are not meant to limit the invention as described herein, which is defined by the claims which follow thereafter.

EXPERIMENTAL DETAILS First Series of Experiments

Polyvalent (Hexavalent) Conjugate Vaccine for Prostate, Breast, Ovarian and Small Cell Lung Cancer

Tumor-specific antigens have been identified and pursued as targets for vaccines. The inventors, previous work has shown that monovalent vaccines utilizing the tumor antigens Globo H, Lewis^(y), GM2, glycosylated MUC-1, Tn(c), or TF(c) conjugated to KLH to be safe with local erythema and edema but minimal systemic toxicities. As a result of vaccination with these monovalent vaccines, most patients generated specific high titer IgM or IgG antibodies against the respective antigen-KLH conjugates. The present invention provides a hexavalent vaccine wherein the components of the monovalent vaccines are combined and administered with an adjuvant as treatment for prostate, breast, ovarian and small cell lung cancer.

A vaccine consisting of a unique combination of six tumor antigens administered with a saponin immunological adjuvant QS-21 or GPI-0100. The antigens are glycosylated MUC-1-32mer, Globo H, GM2, Le^(y), Tn (c), and TF(c). In each case the antigen is conjugated to Keyhole Limpet Hemocyanin (KLH).

The preferred ranges of the antigen and adjuvant doses are as follows:

-   -   Glycosylated MUC-1-32mer: 0.1 to 30 μg;     -   Globo H: 0.1 to 100 μg;     -   GM2: 0.1 to 100 μg;     -   Le^(y): 0.1 to 60 μg;     -   Tn(c): 0.1 to 10 μg;     -   TF(c): 0.1 to 30 μg;     -   QS-21: 100 μg;     -   GPI-0100: 1 or 25 mg.

Example 1 A Phase I Multivalent Conjugate Vaccine Trial for Patients with Biochemically Relapsed Prostate Cancer

1.0 PROTOCOL SUMMARY

2.0 OBJECTIVE

3.0 BACKGROUND AND RATIONALE

4.0 VACCINE PREPARATION

5.0 IMMUNIZATION SCHEDULE

6.0 PRE- AND POST-THERAPY EVALUATION

7.0 RESPONSE CRITERIA

8.0 BIOSTATISTICAL CONSIDERATIONS

9.0 REFERENCES

1.0 Protocol Summary:

This is a phase I pilot trial designed to assess safety using a multivalent conjugate vaccine. This trial is based on the results of eight dose-seeking phase I monovalent glycoprotein and carbohydrate conjugate vaccine trials which have been shown to be consistently immunogenic in man. These trails also allowed us to screen candidate antigens for their ability to generate high titer specific antibodies against the immunizing antigen. This vaccine will consist of the highest dose of synthetic glycoprotein and carbohydrate antigens shown to elicit high titer IgM and IgG antibodies in patients with biochemically relapsed prostate cancer. The inventors' previous work has shown the monovalent vaccines to be safe with local erythema and edema but minimal systemic toxicities. Among the antigens to be included in the multivalent vaccine are carbohydrate antigens Globo H and GM2 and the glycoprotein antigens glycosylated MUC-1-32mer, Lewis^(y), Tn(c), and TF(c). The patient populations to be targeted are those patients who have failed primary therapies such as prostatectomy or radiation or have been on intermittent hormonal therapy and have remained hormonally sensitive in the absence of radiographic disease. These populations must have as the sole indication of disease progression, a rising PSA. The inventors' data from approximately 160 men who participated in earlier monovalent vaccine trials against the aforementioned antigens have shown that a treatment effect in the form of a decline in PSA log slopes compared with pretreatment values could be seen in patients with minimal tumor burden. A phase III double blind randomized trial with two hundred forty patients is planned based on the safety data accrued form this proposed phase I trial. The primary endpoint of the study will be the ability to assess the safety of the vaccine and the humoral response to a multivalent conjugate. Secondary endpoints will be to evaluate post-therapy changes in PSA.

2.0 Objectives:

2.1 The primary endpoints of the study are:

2.1.1 To determine the safety of a multivalent conjugate vaccine in patients with prostate cancer who have biochemically relapsed following primary therapies such as surgery or radiation.

2.1.2 Measure the antibody response against the individual components of the vaccine and to correlate the response to subsequent clinical course.

2.2. The secondary endpoints will be:

2.2.1 To assess post-immunization changes in prostate specific antigen levels and other objective parameters of disease (radionuclide bone scan and/or measurable disease if present.

3.0 Background and Rationale:

3.1 Prostate cancer: Over 180,000 cases of prostate cancer will be diagnosed in the United States in 2000.¹ Of these, 30-35% will present with tumors beyond the confines of the gland, while an additional 25% will develop metastases in the course of the disease despite local therapies. In these cases, a rising PSA antedates the development of overt metastases by a median of 12-24 months. Androgen ablation is the standard treatment with upwards of 70% of cases showing a normalization of an abnormal PSA after therapy. When to initiate treatment remains an area of controversy and there is no evidence that deferring therapy compromises outcomes. This observation, coupled with the fact that most patients relapsed within a median of 12-18 months², and that most men can not tolerate the side effects of castration including impotency, weight gain and hot flashes, has led to the search for alternative therapies. One such approach involves enhancing the body's own immune system as a means to treat local disease and prevent disease progression. PSA monitoring allows the identification of patients with low-volume disease, in whom an immunostimulatory approach may be more efficacious relative to a heavily pretreated, symptomatic population with large tumor burdens. Vaccinations represent a safe intervention with minimal toxicities that can be given as an adjuvant to surgery or radiation therapy in men at risk for systemic relapse. They can also be offered to men with minimal tumor burdens who are progressing and who are not willing to accept toxicities of hormonal therapy or chemotherapy. Because hormonal status may effect antigen expression and regulation, we propose to enroll patients with different hormone sensitivities. This will include patients who have not received hormonal therapy or have been on intermittent hormonal therapy.

3.2 PSA as an endpoint for clinical trials: The availability of serum PSA determinations provides a unique trial design for testing new therapies rapidly as changes in PSA levels over time correlate well with clinical outcomes.³ This relationship holds for both hormone-naïve and hormonally relapsed disease. Once sequential elevations in PSA are documented in the setting of castrate testosterone levels, clinical symptoms develop in a median of 3-6 months. This observation justifies treatment in the setting of rising PSA values, using post-therapy changes in PSA as the outcome measure. With this design, therapeutic approaches that do not produce a defined degree of decline in PSA on multiple determinations for a defined duration (vide infra) are not evaluated further.²

3.3 Immunologic approaches: Augmentation of the immune response to cancer can be attempted by two basic approaches: non-specific immunopotentiation which constitutes the bulk of past and current efforts at cancer immunotherapy, and specific immunization which has not really been evaluated in the treatment of cancer but has contributed much to the control of infectious diseases. It is the knowledge of microbial antigens which has permitted the development of successful specific immunization against infections. The lack of availability of well-defined human cancer antigens, on the other hand, has prevented exploration of specific immunization in the context of cancer as it should be explored, using vaccines of defined cancer-restricted antigenicity and demonstrating their immunogenicity in cancer patients.

3.3.1 The role of carbohydrates and mucins in prostate cancer: Carbohydrate antigens have proven to be clinically relevant and (aside from vaccines against toxins) are the only defined bacterial antigens used in vaccines against bacterial pathogens. Immunization with carbohydrate antigens has also resulted in directed antibody responses against human tumor cells (reviewed in 4) presumably because these antibodies are known to mediate antibody-dependent cell-mediated and complement mediated lysis of tumor cells, complement-induced inflammation, and phagocytosis by the reticulo-endothelial system. The inventors' previous study in prostate cancer focused on defining the antigens expressed on the surface of prostate cancer cells.

3.3.2 Results with eight monovalent phase I trials using glycoprotein peptides and carbohydrate antigens. Immunohistochemistry using well-defined monoclonal antibodies against glycoprotein and carbohydrate antigens have shown that primary and metastatic prostate carcinoma specimens express these heretofore unknown antigens and that these molecules can serve as targets for immune recognition. We have studied two mucin peptide antigens, MUC-1 and MUC-2 conjugated to KLH and given with the immune adjuvant, QS21 in the phase I setting as a dose escalating trial with 10, 30 100 and 3 μg. Patients received five subcutaneous vaccines over the course of twenty-six weeks at weeks 1,2, 3, 7, and 19. Twenty patients were treated in the MUC-1-KLH-QS21 trial and fifteen were treated with MUC-2-KLH-QS21 trial. All patients developed high titer IgM and IgG antibodies specific for the immunizing peptide. Antibody titers rose by week 7 and declined usually by week 19, the time of the fifth and final vaccine. Unexpectedly, a treatment effect was observed after the vaccine trial was completed in the form of a declining PSA log slope compared with pretreatment values in approximately two-thirds of patients. In many patients, the slope began to show a decline by week 38 with subsequent declines by week 60. The initial decline corresponded to the rise of antibodies following the last immunization received at week 19. Five patients who were treated with the MUC-1-KLH conjugate in 1996 continue to have stable PSA log slopes without radiographic evidence of disease. Patients who were treated with MUC-2-KLH conjugate also demonstrated a similar treatment effect, however, the trial has not as yet reached maturity. The vaccines were found to be safe with erythema, tenderness and edema at the injection site. No evidence of autoimmunity or systemic toxicity was observed.

3.3.3 Experience with glycolipid and carbohydrate antigens. Eighteen patients have undergone immunization with Globo H, a glycolipid antigen expressed on prostate cancer cells. This is the first purely synthetic complex carbohydrate antigen used for immunization in man capable of generating high titer specific antibodies (median peak titer 1:320, IgG median titer 160) capable of mediating complement lysis of tumor cells. Several patients generated IgM antibody titers of 1:20,480). Patients were immunized with 10, 30 100 or 3 μg of Globo H-KLH plus QS21 over twenty-six weeks. Of the patients immunized with this vaccine, six remain active with stable PSA log slopes and no radiologic evidence of evidence of disease over the last 2½ years. This vaccine was found to be safe with no evidence of systemic toxicity. Ganglioside antigens (acidic glycosphingolipids expressing sialic acid at one end and ceramide at the other) was also investigated in a trial comparing the immunogenicity of higher doses of QS21. Using GM2-KLH at 30 μg, a dose previously established in melanoma trials, 18 patients were immunized with either the GM2 conjugate plus QS21 at the standard dose of 100 μg or QS21 at 225 Hg. Because of its potential for systemic toxicity, the latter vaccine was given as three separate immunizations to three separate sites as GM2-KLH at 10 μg plus QS21 at 75 μg subcutaneously. No difference in antibody titers were observed in two groups of patients; although two patients from the group given the higher dose of QS21 experienced grade II myalgias. Several patients also exhibited a decline in PSA log slopes but there did not appear to be any difference between groups with regard to treatment effects.

4.0 Vaccine Preparation

Globo H, Lewis^(y), Tn(c), TF(c) are synthesized in the laboratory of Bio-Organic Chemistry headed by Dr. Samuel Danishefsky. MUC-1-32mer is synthesized in the Core Peptide Synthesis Facility of The Rockefeller Research Laboratories under the aegis of Dr. Paul Tempst. It is glycosylated with Tn by Dr. Henrik Clausen at the University of Copenhagen, Copenhagen, Denmark. GM2 is extracted from rabbit brains by Progenics, Inc., Tarrytown, N.Y.

4.1 Globo H. MUC-1-32mer, GM2, Lewis^(y), Tn(c) and TF(c)-KLH conjugation: The above antigens will be covalently attached to KLH in Dr. Livingston's laboratory. Antigen-KLH ratios between 150/1 and 800/1 assuming a KLH molecular weight of 5×10⁶ will be accepted. Gels will be performed and western blot analysis will be conducted with each lot of antigen-KLH for comparison to future lots. Sterility and safety testing with each lot plus QS21, at >50 times the dose/meter² to be used in clinical trials will be performed. No growth in culture and no adverse reaction in mice or Guinea pigs (including weight loss of 10% or more) will be tolerated. Two or more mice will be immunized with each antigen-KLH batch on 2-3 occasions at 1-2 week intervals and post immunization sera tested. Antibody titers of 1/200 or greater against antigen and 1/40 by IA or FACS staining of >25% of antigen positive cells will be accepted as proof that the construct has the appropriate immunogenicity.

4.2 Antigen Doses: Based on previous vaccine trials in prostate cancer patients, the following doses have been established for the multivalent trial: glycosylated MUC-1-32mer, 3 μg; Globo H, 10 g; GM2, 10 μg; Le^(y), 10 μg; Tn(c), 3 μg; and TF(c), 3 μg. QS21 will be used at 100 μg as no significant difference in immunogenicity was observed with doses as high as 225 μg.

4.3 Safety testing: Samples from the materials are sent for sterility and safety testing. Immunogenicity of the individual peptides/carbohydrates have been previously confirmed in mice.

5.0 Immunization Schedule;

5.1 Patient selection: All patients with evidence of biochemical relapse will be considered. Hormonal status will be recorded on the basis of serum testosterone levels as follows: Patients who have progressed after primary surgery or radiation (with or without neo-adjuvant androgen ablation) who have non-castrate levels of testosterone (>50 ng/ml) will be eligible.

5.2 Interval: The immunization schedule that we will utilize was derived from the inventors' studies with other glycoprotein and carbohydrate conjugate vaccines in patients with melanoma, colon and breast cancers.

5.3 Treatment schedule and dose: Fifteen patients will be treated with specified doses of each carbohydrate or peptide constituent as has been determined previously based on earlier monovalent trials completed. QS21 will be administered at the standard dose of 100 ug. Sites: The vaccine conjugate will be administered subcutaneously to random sites on the upper arms and upper legs.

5.4 Dose modifications: If a patient experiences a Grade III or greater local or Grade II or greater systemic toxicity at any time a decrease by 50% in all components of future vaccinations will be administered for that patient.

6.0 Pre- and Post-Therapy Evaluation:

6.1 Outcomes: The study evaluation will include parameters to assess the safety of the vaccine, antitumor effect, as well as assessments of immune function. Interval safety assessments will include the Patient Diary. An overall antitumor assessment will be performed during weeks 13 and 26. If the patient has not demonstrated progression of disease at week 13 or 26, he will continue on protocol. Upon completion of the trial, he will be monitored every 3 months with bloodwork and imaging studies for the next 2 years or until disease progression.

6.2 Safety and antitumor effects:

STUDY WEEK O^(a,c) 1 2 3 7 9 13 19 26 Clinical: Performance Status X^(b) — — X X X X X X^(h) Interval Hx & PE X^(b) — — X X X X — X CBC, Diff, Plt. X — — X X — X — X CMP^(d), LDH X — — X — — X — X Uric acid, Phosphorus X — — X — — X — X Prothrombin time X — — — — — — — — PSA, Ac. Phos X — — X X — X — X Testosterone X — — — — — — — — U/A X — — X X X X — X Stool guaiac X — — — X — — — X Pathology Review^(e) X — — — — — — — — Imaging:^(f) Chest X-ray X — — — — — X — X Bone scan X — — — — — X — X CT Scan or MRI X — — — — — X — X Overall response — — — — — — X — X assessments^(g) Consent for Pathologic X Correlates^(i) ^(a)Baseline studies prior to immunization. ^(b)Within 7 days of the first immunization. ^(c)Within 15 days of starting treatment for biochemical studies; 30 days for imaging studies. Includes total bilirubin, SGOT, LDH, Alkaline Phosphatase, Creatinine, BUN. ^(d)CMP Includes total bilirubin, SGOT, ALT, Sodium, Potassium, Chloride, CO₂, Calcium, Glucose, Total Protein, Abumin, Alkaline Phosphatase, Creatinine, BUN. ^(e)Patients will be asked to obtain tissue blocks from previous diagnostic/therapeutic procedures will be obtained and the patient's tumor evaluated for the presence of the antigens by immunohistochemistry. The presence of any antigen on paraffin material is not a criterion for entry and no biopsy procedures will be performed specifically for enrollment. ^(f)Abdominal and pelvic CT scans with and without contrast, chest x-ray and any other tests deemed necessary to document evaluable disease. ^(g)Overall response assessment includes the repetition of abnormal imaging and biochemical studies used to assess disease, and in selected cases, immune function. ^(h)Repeat at 3 month intervals for 2 years or until disease progression is documented. ^(i)Patients will be asked to sign a separate consent for pathologic correlative studies under IRB [90-40: Dr. H. Scher, P.I. - Molecular correlations in human prostate cancer].

6.3 Immune function:

STUDY WEEK 0 1 2 3 7 9 13 19 26 VACCINATION* — 1 2 3 4 — — 5 — B-CELL TESTING — 1 2 3 4 5 6 7 8 *No skin tests will be performed as previous trials indicated that there is minimal or no reactivity with intradermal administration of the antigens studied.

Antibody response: Peripheral blood (30 cc) will be drawn prior to vaccine immediately before each vaccination, as well as weeks 9, 13, and 26 to assess B-cell function. Thereafter, blood will be drawn at 3-month intervals (up to one year from the first vaccination), or as long as detectable immunity against the antigens persist. Depending on the antibody response, additional testing involving proliferation and cloning may be performed at a later date. The patients' sera will be tested by ELISA for antibodies against purified antigens as well as a variety of cell lines expressing (or not) the antigens included in the vaccine.

7.0 Response Criteria:

7.1 Patients WITHOUT bi-dimensionally measurable disease are evaluable by post-therapy changes in PSA as follows:

7.1.1 Complete Response (CR): Normalization of the PSA (≦

1.0 or 2.0 as defined in 4.1.1) for 3 successive evaluations at least 2 weeks apart.

7.1.2 Partial Response (PR); Decrease in PSA value by >50% above baseline (without normalization) for 3 successive evaluations.

7.1.3 Stabilization (STAB): Patients who do not meet the criteria for PR or PROG for at least 90 days will be considered stable.

7.1.4 Progression (PROG): Three consecutive increases in PSA, to >50% above baseline.

7.2 Duration of response: Non-measurable disease: Time from initiation of therapy until a 50% increase from the PSA nadir value is documented on three successive determinations.

8.0 Biostatistical Consideration

8.1 This is an exploratory study to study the safety of a multivalent conjugate vaccine which will be taken to phase III clinical trials. Patients with prostate cancer who have experienced a PSA recurrence after radical prostatectomy or radiation therapy are eligible. All fifteen patients will receive the same dose. The dose is based on previous vaccine trials in prostate cancer patients, the following doses have been established for the multivalent trial: glycosylated MUC-1-32mer, 3 μg; Globo H, 10 μg; GM2, 10 μg; Le^(y), 10 μg; Tn(c), 3 μg; and TF(c), 3 μg. QS21 will be used at 100 μg as no significant difference in immunogenicity was observed with doses as high as 225 μg. Subjects will be followed for two years or until the development of metastatic disease. Bone scan and CT scans (or MRI where clinically appropriate) will be performed approximately at week 13, 26, and approximately every 3 months thereafter until the development of metastatic disease. In addition, PSA measurements will be obtained at weeks 3, 7, 13, 26, and every approximately 3 months thereafter in order to study the effect of the vaccine on the probability of developing metastatic disease and the effect of the vaccine on PSA slope over time, respectively.²⁴

8.2 In order to be eligible for the study, patients must have a rising PSA following radical prostatectomy or radiation therapy. This detection of PSA following treatment must occur within two years. Using the ASTRO definition, three consecutive PSA rises are considered a biochemical failure after radical prostatectomy or radiation therapy. The date of failure should be the midpoint between the postsurgical (or postirradiation) nadir PSA and the first of the three consecutive rises.²⁵ In addition, patients must have a PSA doubling time (DT) less than 5 months. PSA doubling time is determined prior to treatment and is equal to ln(2) divided by the least squares derived slope of log PSA over time (log PSA slope>0.15).26 The time interval in which PSA DT will be based will consist of a minimum of three PSA measurements in a twelve-month interval prior to randomization. Patients who meet this requirement are considered at a higher risk for metastatic disease and will be eligible for this trial.

8.3 The primary objective of this study is to determine the safety and the humoral response of the multivalent vaccine in preparation for the phase III trial. The primary endpoint will be the time to radiographic progression of disease. The secondary objective is to study the rate of change in PSA over time.

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-   1. Wingo, P A, Tong, T, Bolden S: Cancer statistics, 1995. Ca Cancer     J Clin, 45:8-30, 1999. -   2. Scher, H I, Cordon-Cardo C: Current and future therapeutic     strategies in metastatic hormonal resistant prostate cancer: therapy     based on phenotype. Problems in Urology, 7:226-253, 1993. -   3. Scher H I: Prostatic cancer: where do we go from here? Current     Opin Oncol, 3:568-574, 1991. -   4. Livingston, PO: Augmenting the immunogenicity of carbohydrate     tumor antigens. Semin Cancer Biol, 6:357-366, 1995. -   5. Itzkowitz S H, Bloom E J, Kokai W A, Modin G, et al: Sialosyl Tn:     A novel mucin antigen associated with prognosis in colorectal cancer     patients. Cancer 66:1960-1966, 1990. -   6. Burchell, J, Taylor-Papadimitriou J, Boishell M, Gendler, S et     al: A short sequence, with the amino acid tandem repeat of a     cancer-associated mucin, contains immunodominant epitopes. Int J     cancer 44:691-696, 1989. -   7. Gendler S, Lancaster C, Taylor-Papadimitriou J, Duhig T, et al:     Molecular cloning and expression of human tumor-associated     polymorphic epithelial mucin. J Biol Chem 265:15286-15293, 1990. -   8. Gendler S J, Spicer A P, Lalani E-N, et al: Structure and biology     of a carcinoma-associated mucin, MUC1. Am Rev Res, 144:S42-S47,     1991. -   9. Carrato C, Balague C, De Bolos C, Gonzalez E, Gambus G. et al:     Differential apomucin expression in normal and neoplastic human     gastrointestinal tissues. Gastroenterology, 107:160-172, 1994. -   10. Ho S B, Niehans G A, Lyftogt C, Yan P s, et al: Heterogeneity of     mucin gene expression in normal and neoplastic tissues. Cancer Res,     53:641-651, 1993. -   11. Slovin, S. F., Livingston, P., Zhang, S., Keeperman, K. Mhatre,     S., Adluri, S., Ghossein, R., Glazewski, J., Gordils, J.,     Danishefsy, S., and Scher, H. I.: Targeted therapy in prostate     cancer (PC): vaccination with a glycoprotein, MUC-1-KLH-QS21 peptide     conjugate. Proc Am Soc Clin Onc., 16:311a (Abstr 1107), 1997. -   12. Slovin, S. F., Livingston, P., Keeperman, K., Mhatre, S.,     Danishefsky, S., Glazewski, J., Adluri, S., Zhang, S., Gordils, J.,     and Scher, H. I.: Targeted therapy in prostate cancer: vaccination     with glycoprotein, MUC-1-KLH-QS21 peptide conjugate. J. Urol.,     157:160 (Abstr 620), 1997. -   13. Zing P X, Apostolopoulos V, Prenzoska J, et al: Petioled binding     sites recognized by anti-mucin (MUC-2) antibodies. Scand J Immun,     591-592, 1994. -   14. Slovin, S. F., Ragupathi, G., Donaldson, C., Olkiewicz, K.,     Terry, K., DePaolo, R., Livingston, P. O, and Scher, H. I.:     MUC-2-KLH conjugate vaccine: Immunogenicity in patients with     relapsed prostate cancer. Proc. Amer. Assoc. Cancer Res,     40:312(Abstr#2071), 1999. -   15. Ragugpathi, G., Adluri, R., Amaravathi, R., Howarad, L.,     Gilewski, T., Slovin S. F., and Livingston, P. O.: Specificity     analysis of sera from breast and prostate cancer patients vaccinated     with MUC1-KLH and MUC-2-KLH conjugate vaccines. Proc. Amer. Assoc.     Cancer Res, 40:312(Abstr #2070), 1999 -   16. Slovin, S. F., Livingston, P. O., Danishefsky, S., Mhatre, S.,     Ragupathi, R., Depaolo R., Sames, D., Terry K., Bauso, A., Kelly, W.     K., Fazzari, M., and Scher, H. I.: Carbohydrate vaccines as     immunotherapy for prostate cancer (PC): Globo-H-KLH conjugate plus     QS21. Proc Am Soc Clin Onc, 17:433a (Abstr 1669), 1998. -   17. Slovin, S F, Ragupathi G, Adluri S, Ungers G, Terry K, Kim S,     Spassova M, Bornmann, W G, Fazzari M, Dantis L, Olkiewicz K, Lloyd K     O, Livingston P O, Danishefsky S J, and Scher H I: Carbohydrate     vaccines in cancer: Immunogenicity of a fully synthetic globo     hexasaccharide conjugate in man. Proc Natl Acad Sci, USA,     96:5710-5715, 1999. -   18. Slovin, S., Ragupathi, G., Israel, R., Terry, K., Bauso, A.,     Fazzari, M., Kelly, K., Reyes, S., Livingston, P., and Scher, H.     Ganglioside vaccines in relapsed prostate cancer (PC): Experience     with GM2-KLH conjugate plus the immunologic adjuvant, QS21-A trial     comparing QS21 doses. Proc. Amer. Soc. Clin. Onc., 18:316 Aa(Abstr     #1214), 1999. -   19. Livingston P O, Natoli E J, Calves M J: Vaccines containing     purified GM2 ganglioside elicit antibodies in melanoma patients.     Proc natl Acad Sci USA, 27:537, 1987 (Abstract). -   20. Slovin, SF, et al: Tn-cluster (c)-KLH/PAM vaccine conjugates in     biochemically relapsed prostate cancer (PC): Phase I trial results.     Proc. Amer. Assoc. Cancer Res, in press. -   21. MacLean G D, Reddish M, Koganty, R R, Wong T, Gandhi S,     Smolenski M, Samuel J, Nabholtz J M, and Longenecker B M:     Immunization of breast cancer patients using a synthetic sialyl-Tn     glycoconjugate plus Detox adjuvant. Cancer Immunol Immunotherap     36:215-222, 1993. -   22. Zhang S, Walberg L A, Ogata S, Itzkowitz S H, Koganty R R,     Reddish M, Gandhi S S, Longenecker B M, Lloyd K O and Livingston P     O: Immune sera and monoclonal antibodies define two configurations     for the sialyl Tn tumor antigen. Cancer Res 55:3364-3368, 1995. -   23. MacLean G D, Bowen-Yacyshyn M B, Samuel J, Meikle A, Stuart G,     Nation J, Poppema S, Jerry M, Koganty R, Wong T, and Longenecker B     M: Active immunization of human ovarian cancer patients against a     common carcinoma (Thompson-Friedenreich) determinant using a     synthetic carbohydrate antigen. J Immunotherapy, 11:292-305, 1992 -   24. Scher, H. I, Slovin, S. F., Kelly, W. K., Livingston, P. O.,     Danishefsky, S., Fazzari, M., Terry, K. And Heller, Glen:     Intermediate markers in assessing response to vaccine therapies.     Proc Am Soc Clin Onc, 17:324a (Abstr 1247), 1998. -   25. Patel, A, Dorey, F, Franklin, J, and DeKernion, J B: Recurrence     pattterns after radical retropubic prostatectomy: Clinical     usefulness of prostate specific antigen doubling times and log slope     prostate specific antigen. J Urol, 158:1441-1445, 1997. -   26. O'Brien, PC, Fleming, T R: A multiple testing procedure for     clinical trials. Biometrics 35:549-S56, 1979.

Example 2 Hexavalent Vaccine Immunogenicity Trial in Mice

Methods

Serological Analyses

1. ELISA (Enzyme-Linked Immunosorbent Assay):

ELISA assays were performed as described below. Antigen in ethanol or in 0.1 M carbonate buffer (pH 11) were coated on ELISA plates at 0.2 μg/well for glycolipids and 0.1 g/well for peptides. Serially diluted antiserum was added to each well and alkaline phosphatase-conjugated goat anti-mouse IgM or anti-mouse IgG was added at a dilution of 1:200 (Southern Biotechnology Associates, Inc, Birmingham, Ala.). Goat anti-mouse IgG and IgM conjugated with alkaline phosphatase obtained from Kierkegaard and Perry Labs, (Gaithersburg, Md.) were used as second antibodies. ELISA titer is defined as the highest dilution yielding an absorbance of 0.1 or greater over that of normal control mouse sera.

2. Cell Surface Reactivity Determined by FACS:

The cell surface reactivity of immune sera was tested on human cell lines. Single cell suspensions of 2×10⁵ cells/tube were washed in PBS with 3% fetal calf serum (FCS) and 0.01M NaN₃ and incubated with 20 μl of 1:20 diluted sera or monoclonal antibody mAb for 30 min on ice. After two washes with 3% FCS in PBS, 20 μl of 1:15 diluted goat anti-mouse IgM or IgG-labeled with fluorescein-isothiocyanate (FITC, Southern Biotechnology Associates Inc. Birmingham, Ala.) was added, and the mixture incubated for 30 min. After a final wash, the positive population and mean fluorescence intensity of stained cells were differentiated using FACScan, Becton & Dickinson Immunocytometry, San Jose, Calif.

Appendix B

Hexavalent Vaccine immunogenicity Trial in Mice Four female CB6F1 mice were vaccinated weekly for three weeks with Hexavalent vaccine (Hexavalent vaccine in Polyval-KLH conjugate plus 20ug QS21 per mouse). The injections were SC, at 2 sites, with 95ul/site. [Vial labeled Polyval in Polyval-KLH conjugate plus 100ug QS21/1.0 ml. Total vol. 1.0 ml. Lot # 081100] Pre-vaccination sera was drawn from each mouse. Sera was drawn again at 10 days post third vaccination. The mice were weighed prior to and post vaccination (at 24 hr post, at 48 hr post, at one week post and at 2 weeks post). For controls, the following monoctonal antibodies were used: VK9 anti-Globo-H BR96 anti-Le^(y) HMFG1 anti-MUC1 αTn Ab anti-Tn αGM2 Ab anti-GM2 49H.8 anti-TF SEROLOGY ELISA plates were coated with 0.1 ug/well of one of the following antigens: GloboH-ceramide, GM2(IgM), MUC1G5, Tn-′HSA, Tf-′HSA ELISA plates were coated with 0.2 ug/well of one of the following antigens: GM2 (IgG). Ley Sera was tested at an initial dilution of 1:40, with subsequent 2-fold dilutions (with the exception of GloboH for which 3-fold dilutions were used). FACS analysis was performed on two cell lines: MCF7 and LSC (5 × 10⁸ cells per tube). Sera was added at a 1:20 dilution (25 ul/tube). Each post 3rd vacc. sera was set against its corresponding pre-sera (each pre-sera was set at 10%). Appendix B Results ELISA Globo H GM2 Le^(Y) IgG IgM IgG IgM IgG IgM Mouse # presera post 3rd presera post 3rd presera post 3rd presera post 3rd presera post 3rd presera post 3rd 1 0 40 120 1,080 40 40 40 80 0 40 80 640 2 0 80 40 3,240 40 40 40 80 0 40 80 80 4 0 360 120 3,240 0 0 0 80 0 40 40 640 5 0 40 0 3,240 0 40 0 80 0 160 40 320 +control VK9 1:25,600 αGM2 >>>1:1,000,000 BR96(1ug/ul) 1:3,200 MUC1G5 Tf-′HSA Tn-′HSA IgG IgM IgG IgM IgG IgM Mouse # presera post 3rd presera post 3rd presera post 3rd presera post 3rd presera post 3rd presera post 3rd 1 0 10,240 0 80 0 640 0 80 0 1,280 0 80 2 0 20,480 0 320 0 640 0 160 0 640 0 5,120 4 0 5,120 0 40 0 5,120 0 160 0 10,240 0 640 5 0 10,240 0 160 0 10,240 0 160 0 10,240 40 640 +control 49H.8 1:1,500 aTn 1:25,600 FACS MCF7 LSC IgG IgM IgG IgM Mouse # presera post 3rd presera post 3rd +controls presera post 3rd presera post 3rd +controls 1 10.02% 95.76% 11.52% 89.79% VK9 1.13% 10.11% 42.28% 9.63% 58.23% VK9 0.95% 2 10.49% 95.30% 10.48% 95.61% BR96 97.11% 9.60% 28.88% 10.75% 93.47% BR95 94.85% 4 9.78% 94.71% 11.36% 95.96% HMFG1 62.31% 9.93% 27.09% 11.12% 96.28% HMFG1 1.19% 5 9.78% 95.48% 9.86% 94.49% αTn Ab 78.62% 10.59% 23.46% 10.16% 93.23% αTn Ab 57.63% 2° Ab alone 1.28% 1.15% αGM2 Ab 94.91% 1.18% 1.14% αGM2 Ab 63.94% 49H.8 0.14% 49H.8 0.26%

Second Series of Experiments

Polyvalent (Heptavalent) Conjugate Vaccine for Prostate, Breast, Ovarian and Small Cell Lung Cancer

Tumor-specific antigens have been identified and pursued as targets for vaccines. The inventors' previous work has shown that monovalent vaccines utilizing the tumor antigens Globo H, Lewis^(y), GM2, glycosylated MUC-1, Tn(c), sTn(c), or TF(c) conjugated to KLH to be safe with local erythema and edema but minimal systemic toxicities. As a result of vaccination with these monovalent vaccines, most patients generated specific high titer IgM or IgG antibodies against the respective antigen-KLH conjugates. The present invention provides a heptavalent vaccine wherein the components of the monovalent vaccines are combined and administered with an adjuvant as treatment for prostate, breast, ovarian and small cell lung cancer.

A vaccine consisting of a unique combination of seven tumor antigens administered with a saponin immunological adjuvant QS-21 or GPI-0100. The antigens are glycosylated MUC-1-G5, Globo H, GM2, Le^(y), Tn(c), sTn(c), and TF(c). In each case the antigen is conjugated to Keyhole Limpet Hemocyanin (KLH).

The preferred ranges of the antigen and adjuvant doses are as follows:

-   -   Glycosylated MUC-1-G5: 0.1 to 30 μg;     -   Globo H: 0.1 to 100 μg;     -   GM2: 0.1 to 100 μg;     -   Le^(y): 0.1 to 60 μg;     -   Tn(c): 0.1 to 100 μg;     -   sTn(c): 0.1 to 100 μg;     -   TF(c): 0.1 to 30 μg;     -   QS-21: 25-200 μg;     -   GPI-0100: 1-25 mg.

Example 1

Phase I clinical trial protocol using the heptavalent vaccine

Example 2

Heptavalent vaccine immunogenicity trial in mice

-   -   1. Methods     -   2. Results

Example 1 Pilot Phase I Trial in Patients with Epithelial Ovarian, Fallopian Tube, or Peritoneal Cancer with a Polyvalent Vaccine-KLH Conjugate+Qs-21

1.0 PROTOCOL SUMMARY

2.0 OBJECTIVE

3.0 BACKGROUND AND RATIONALE

4.0 VACCINE PREPARATION

5.0 TREATMENT SCHEDULE AND DOSE

6.0 EVALUATION DURING STUDY

7.0 BIOSTATISTICAL CONSIDERATIONS (Endpoints)

8.0 BIBLIOGRAPHY

1.0 Protocol Summary and Program Plan

Patients with epithelial ovarian, fallopian tube, or peritoneal cancer who receive surgical cytoreduction and platinum/taxane containing chemotherapy have a significant chance of entering complete clinical remission but unfortunately approximately 70% will eventually relapse. These patients in clinical remission have minimal residual disease, and are excellent candidates in which to evaluate novel consolidation strategies in an attempt to improve outcome. This pilot polyvalent protocol represents the culmination of a series of monovalent phase I vaccine trials at the center demonstrating the immunogenicity of the various component antigens. It represents the transition between the phase I monovalent trial program in second remission, to the planned development of larger trials designed to evaluate efficacy. Immunization with the individual antigens selected for this vaccine has been consistently immunogenic in the majority of patients. No confirmed systemic toxicity has occurred related to vaccine administration. It is expected that the immunogenicity will remain unchanged, and that no systemic toxicity will occur with polyvalent vaccine administration. Eligible patients for this pilot trial are those patients initially with stage II-IV disease in complete clinical remission following primary therapy, or following relapse and re-induction to remission with additional chemotherapy. In this trial, patients will receive an antigen defined vaccine with the following ganglioside components: a) GM2, b) Globo-H; the blood group related antigens: c) TF(c), d) s-Tn (c), e) Tn (c) f) Lewis-Y; and g) the protein antigen MUC-1-G5 (glycosylated). The primary endpoints of this pilot study are safety, and confirmation of continued immunogenicity. The secondary endpoint will be to characterize the nature and duration of the antibody response.

2.0 Objectives

2.1 The primary endpoints of this pilot study are to determine the safety of polyvalent vaccine administration, and continued immunogenicity in patients prior to conducting a large, randomized study.

2.2 The secondary endpoint is to further characterize the nature and duration of the antibody response generated by the polyvalent vaccine (ELISA and FACS)

3.0 Background and Rationale

3.1 Disease Background and Suitability for Treatment

In 1999, approximately 22,500 new cases of ovarian cancer were diagnosed, and it is estimated that 14,500 women died of the disease. Seventy-five percent of patients with ovarian cancer will have spread beyond the ovary at diagnosis. Standard primary treatment consists of cytoreductive surgery followed by a platinum and paclitaxel containing chemotherapy regimen.¹ Many patients have no clinically measurable disease at the end of primary treatment. A review of second-look laparotomy, however, indicates that less than 50% of patients are actually free of disease.² Furthermore, nearly half of patients with a negative second look procedure are destined to relapse and require additional treatment.^(3,4) Overall, only approximately 30% of patients remain disease free with currently available treatment. Given the minimal disease burden at the completion of primary therapy, these patients are ideal candidates in which to evaluate immune modulating strategies.

3.2 Rationale for Polyvalent Vaccines Designed Primarily for Antibody Production

Varied data exists in solid tumors to support the development of immune directed therapy. Studies have emerged in patients with melanoma which demonstrate that naturally acquired 5.6, or actively induced^(7,8) antibodies may improve outcome. In a large clinical trial reported by Reithmuller et al., 189 patients with resected Dukes C colon carcinoma were randomized to receive observation versus postoperative treatment with murine antibody CO17-1A that recognizes the KSA antigen. Toxic effects were limited to infrequent constitutional symptoms. At median follow-up of five years, the death rate was 36% in the treated group versus 51% in the observed group. The advantage of treatment was demonstrated in univariate (p=0.051) and multivariate (p=0.043) analysis when controlling for other known prognostic factors.⁹

The basis for cancer vaccines designed primarily for antibody induction are the many preclinical models demonstrating the ability of passively administered or actively induced antibodies to prevent tumor recurrence¹⁰, the increasing number of clinical trials where passively administered monoclonal antibodies have demonstrated clinical efficacy, and the correlation of antibodies, naturally acquired or vaccine induced, with improved prognosis in several different clinical settings.⁶

EL4 lymphoma naturally expresses GD2 ganglioside, which is recognized by monoclonal antibody 3F8. Vaccines containing GD2 covalently conjugated to KLH and mixed with immunological adjuvant QS21 are the optimal approach to vaccination against GD2. Relatively higher levels of antibody administered two or four days after intravenous tumor challenge or moderate titers induced by vaccine that were present by day two or four after tumor challenge were able to eradicate disease in most mice. If antibody administration was deferred until day seven or ten, little or no benefit could be demonstrated. If the number of cells in the EL4 challenge was decreased, giving a longer window of opportunity, the vaccinations could be initiated after tumor challenge and good protection seen.⁷ These results are consistent with the need to initiate immunization with vaccines inducing antibodies in the adjuvant setting, when the targets are circulating tumor cells and micrometastases. Patients with ovarian cancer in first remission meet these criteria, and unfortunately have a high “event rate” (ie. 80% will relapse) allowing for the rapid assessment of the efficacy of this approach.

The basis for the inventors' emphasis on polyvalent vaccines is tumor cell heterogeneity, heterogeneity of the human immune response and the correlation between overall antibody titer against tumor cells and effector mechanisms such as complement mediated cytotoxicity (CDC) or antibody dependent cell mediated cytotoxicity (ADCC). For example, using a series of 14 tumor cell lines and monoclonal antibodies (mAbs) against 3 gangliosides, investigators at MSKCC have shown that significant cell surface reactivity analyzed by flow cytometry and CDC increased from 2-8 of the cell lines using one of three mAbs to 13-14 of the cell lines when the 3 mAbs were pooled. The median CDC increased 4 fold with the pool of mAbs compared to the best single mAbs.¹¹ Cancers of the ovary express a rich array of cell surface antigens making them especially suitable targets for polyvalent vaccines.

Cell surface antigens (especially carbohydrate cell surface antigens) have proven to be unexpectedly potent targets for immune recognition and attack of human cancers. Many of the more tumor-restricted monoclonal antibodies derived by immunization of mice with human tumor cells have been found to be directed against carbohydrate antigens expressed at the cell surface¹² Immunization against carbohydrate antigens results generally in an antibody response (see references for dissenting views), which is primarily IgM.¹³⁻¹⁵ These antibodies are known to induce CDC, inflammation, and phagocytosis of tumor cells by the reticulo-endothelial system (opsonization).¹⁶ Immunization against cell surface protein antigens can induce a variety of B and T lymphocyte responses. The T lymphocyte responses are difficult to quantify in the context of vaccination trials and are not the focus of this proposal. The antibody responses against protein antigens contain IgM and IgG, both of which can induce complement activation (with regard to IgG depending on the subclass, IgG1 and IgG3 being optimal). IgG antibodies of these subclasses can also induce ADCC.

Antibodies are the primary mechanism for active elimination of circulating pathogens from the bloodstream. They are ideally suited for eradication of free tumor cells and systemic or intraperitoneal micrometastases and they have accomplished this as described above in a variety of preclinical mouse experiments (reviewed in references).^(10,7) In adjuvant immunization trials, the primary targets are individual tumor cells or early micrometastases which may persist for long periods after apparent resection of all residual tumor.¹⁷ After surgery and completion of chemotherapy is the ideal time for immune intervention, and in particular for administration of cancer vaccines aimed at instructing the immune system to identify and kill the few remaining cancer cells. If antibodies of sufficient titer can be induced against tumor antigens to eliminate tumor cells from the blood and lymphatic systems, aggressive local therapies, including surgery, radiation therapy and intralesional treatments might result in long term control of even metastatic cancers.

3.3 Preliminary Studies for the Antigens

GM2 vaccines: Investigators at the center have been refining the ability to induce antibodies against GM2 in melanoma patients for fifteen years, since first demonstrating that vaccines containing purified GM2 could be more immunogenic than vaccines containing tumor cells expressing GM2.18 Initially GM2 adherent to BCG was selected as optimal, inducing IgM antibodies in 85% of patients. This was the basis for a randomized trial comparing immunization with BCG to immunization with GM2/BCG in 122 patients with AJCC Stage 3 melanoma.⁸ The IgM antibodies had a median titer of 1/160 and were short lived (8-12 weeks). IgG antibody induction was rare. Antibody titers have been maintained for over three years by administration of repeated booster immunizations at 3-4 month intervals. When comparing patients as randomized in this trial, no statistically significant difference on overall or disease free survival was seen. Pre-existing GM2 antibodies were seen in 5 patients in the control group, as opposed to one in the GM2 treated group which may have blunted the treatment result. The association between better outcome and the presence of GM2 antibodies was seen (8).

TF, Tn and sTn vaccines: Patients with various epithelial cancers have been immunized with unclustered TF-KLH and sTn-KLH conjugate vaccines plus various adjuvants.¹⁹ High titer IgM and IgG antibodies against TF and sTn antigens have resulted, but we found that the majority of the reactivity detected in sera from immunized mice and patients was against antigenic epitopes present on synthetic constructs which were not present on naturally expressed mucins.²⁰ Based on previous studies with Tn antigen, 21 Kurosaka and Nakada et al. hypothesized that MLS102, a monoclonal antibody against sTn, might preferentially recognize clusters (C) of sTn. In studies at MSKCC with monoclonal antibody B72.3 and with sera raised against TF-KLH and sTn-KLH conjugate vaccines in mice and in patients resulted in the same conclusion.^(20,22) The availability of synthetic TF, Tn and sTn clusters consisting of 3 epitopes covalently linked to 3 consecutive serines or threonines has permitted investigators at MSKCC to prove this hypothesis. In both direct tests and inhibition assays, B72.3 recognized sTn clusters exclusively, and sera from mice immunized with sTn (C)-KLH reacted strongly with both natural mucins and tumor cells expressing sTn.²² Based on this background, we initiated trials with the TF(C)-KLH, Tn(C)-KLH and sTn(C)-KLH conjugate vaccines in patients with breast cancer. Antibodies of relevant high titer specificity, including against OSM or PSM and cancer cells expressing TF, Tn or sTn, have been induced for the first time in the inventors' experience. Based on these results confirming the importance of clustered epitopes and defining their relevant immunogenicity, we are including these clustered antigens in the polyvalent vaccine against ovarian cancer.

Le^(y) and Globo H vaccines: The development of Le^(y) and Globo H vaccines was previously limited by the lack of sufficient quantities of antigen for vaccine construction and testing. Over the last four years, Dr. Danishefsky has successfully synthesized both antigens.^(23, 24) Investigators at MSKCC have immunized groups of mice with Globo H-ceramide plus or minus adjuvants QS-21 and Salmonella minnesota mutant R595, and with Globo H covalently attached to KLH or BSA plus immunological adjuvant QS-21. The highest antibody titers against both synthetic antigen and MCF7 cells expressing Globo H were induced by the Globo H-KLH plus QS-21 vaccine.^(23,24) The antibody titer induced against synthetic Globo H was 1/120,000 by ELISA, the titer induced against MCF7 was 1/320, and potent complement mediated cytotoxicity was seen as well. Le^(y)-BSA and Le^(y)-KLH vaccines have also been tested in the mouse. High titer antibody responses have resulted against the synthetic epitope of Le^(y) and against tumor cells expressing Le^(y) in the majority of mice immunized.²⁵ Based on these results, monovalent phase I clinical trials with Globo H-KLH plus QS-21 and Le^(y)-KLH plus QS21 have been initiated in patients with breast, prostate or ovary cancer. Antibodies against the purified antigens and against tumor cells expressing these antigens were induced in most patients and the manuscript was recently published for the latter.^(26,27)

MUC1 and MUC2 vaccines: Investigators at MSKCC have immunized mice with MUC1-KLH and MUC2-KLH, plus QS-21, and seen induction of consistent high titer IgM and IgG antibodies against MUC1 and MUC2 and human cell lines expressing MUC1 and MUC2, as well as protection from a syngeneic mouse breast cancer expressing human MUC1 as a consequence of gene transduction. Mice were also immunized with vaccines containing MUC1 peptides of various lengths conjugated to KLH by one of three methods or not, and mixed with QS-21 or BCG. MUC1 containing 30 amino acids or more, conjugated to KLH with an MBS bifunctional linker and mixed with immunological adjuvant QS-21 induced the highest titer antibodies.²⁸ Based on these studies in the mouse, a trial was initiated and completed a trial with this MUC1-KLH plus QS-21 vaccine in breast cancer patients who were free of detectable breast cancer after resection of all known disease. Nine patients were treated with a 31 amino acid MUC1 peptide with cysteine at one end for conjugation to KLH and the immune dominant epitope-APDTRPA at the other end.²⁹ No patient had detectable MUC1 serological reactivity by ELISA or FACS prior to immunization. The results are summarized below in table below. Reactivity against MUC1 and tumor cells expressing MUC1 was seen in most patients. A separate group of patients were immunized with MUC2-KLH plus QS21. Analysis of this trial is not yet complete, but the results to date are also summarized in the table below.

The inventors have been unable to demonstrate T-lymphocyte proliferation, interferon γ and IL4 release by ELISPOT assays, CTL activity or positive DTH responses after vaccination with MUC1 or MUC2. The proliferation assays were particularly focussed on in the MUC1 trials. Patients had leukophoresis pre and post vaccination, providing ample lymphocytes for study. After 2 years of steady endeavor, there has been no clear evidence of augmented reactivity against MUC1 peptides of various lengths or, in HLA A2 positive patients against heteroclytic MUC1 peptides with single amino acid changes that increased binding to HLA A2. (personal communication, P.O. Livingston) Pre and post vaccination PBLs from the first 6 patients vaccinated with MUC1 were also sent to the laboratory of Dr. Olivera Finn for CTL precursor frequency analysis. No increase in frequency was seen. Over all, the major difference between results from MSKCC with the 31aa MUC1-KLH plus QS-21 vaccine and Dr. Finn's results with a 104aa MUC1 peptide plus BCG vaccine was that the former had a clearly demonstrable, consistent antibody response, which was reactive with tumor cells. Inhibition assays were performed to better understand this serologic response.³⁰ Much of the IgM response and nearly all of the IgG response were against the immune dominant epitope, APDTRPA, preferentially with RPA at the terminal position.

KSA vaccines: KSA has been prepared in the baculovirus system by Jenner Technologies (San Ramon, Calif.) and 10 mcg/patient has been provided for testing. Due to the small quantity of KSA available, following demonstration of relevant immunogenicity in mice, we treated groups of 9 patients with KSA plus QS21 or with KSA covalently linked to KLH by glutaraldehyde, plus QS21. In neither case was there significant induction of antibodies against KSA that had not been glutaraldehyde treated, or tumor cells expressing KSA. Consequently KSA will not be included in the polyvalent vaccine. The results of this and the other trials with KLH conjugate vaccines are summarized in the table below (personal data, P. O. Livingston).

Summary of Serological Results in Vaccinated Patients Median Median ELISA IgG FACS Median Median Antigen IgM IgG Subclass IgM IgG IA CDC GM2 640 320 IgG1 + 3 +++ ++ ++ ++ Globo H 640 40 IgG1 + 3 ++ + ++ + Lewis^(Y) 80 0 ++ + + + Tn 1280 1280 ++ − + − STn 1280 160 IgG3 +++ − + − TF 320 10 − + − MUC1 1280 5120 IgG1 + 3 + − + − MUC2 2560 2560 pending KSA 40 160 − − − −

Additional variables: Two additional variables have proven critical, the method of conjugation and the epitope ratio of antigen molecules per KLH molecule. The optimal conjugation approached has varied with the antigen. Gangliosides are best conjugated using ozone cleavage of the ceramide double bond and introducing an aldehyde group followed by coupling to aminolysyl groups of KLH by reductive amination. This approach was not as effective for conjugation of Tn, sTn and TF clusters or Globo H to KLH where an M2C2H linker arm has proved most efficient³¹ or for MUC1 or MUC2 where an MBS linker group was optimal.²⁸

The impact of dose and schedule of vaccine administration on antibody response to GM2 vaccines in melanoma patients has also been explored. Immunization 4 times at weekly intervals or biweekly intervals followed by booster immunizations twice at 2-3 month intervals was compared to 6 immunizations at monthly intervals. Initial immunizations at weekly or biweekly intervals resulted in comparable high titers (with high titers occurring slightly sooner at weekly intervals), but remarkably the monthly immunizations resulted in far weaker or undetectable antibody responses in the 6 patients vaccinated.³² GM2-KLH plus QS-21 vaccines prepared at MSKCC and at Progenics Pharmaceuticals have each been tested in dose finding studies such as those proposed in this application. In both cases GM2 doses of 3 ug or less resulted in lower IgM titers and undetectable IgG titers in most patients. GM2 doses of 10, 30 and 100 ug gave comparable IgM and IgG titers.³³ Based an these studies, we have selected the initial weekly schedule of 3-4-immunizations followed by booster immunizations every three months, and the doses for use in the randomized Phase III trial.

3.4 Potential Toxicity of Vaccination

The expected safety of the vaccine is based on the safety of vaccination with the individual components. Clinical experience is growing in clinical trials with vaccine induced antibody responses against each of the included antigens. Antigen expression at secretory borders in these trials, where the majority is located, has induced neither immunological tolerance nor symptomatic autoimmunity once antibodies are present, suggesting they are sequestered from the immune system. Nevertheless, a regular schedule of laboratory studies and physical examinations are designed to detect any abnormalities. This pilot trial will represent one of the first studies to confirm the safety of polyvalent vaccine administration in this setting.

3.5 General Immune Approaches

Various methods have been used to increase the immunogenicity of antigens, and in particular for inducing an IgG response. In preclinical laboratory studies, we have found the covalent attachment of antigen to keyhole limpet hemocyanin (KLH) to be most effective.³⁴ KLH is well tolerated, and has previously caused only mild inflammation at the vaccine injection site. Attachment of KLH may be accomplished by a variety of cross-linking methods. MBS (m-maleimidobenzoly-N-hydroxysuccinimide ester) is the best-known heterobifunctional reagent; and at neutral pH cross-links thiol groups with amino groups. The linkage proceeds via two separate reactions, thus limiting bonds between identical molecules. In addition to linking antigen to immunogenic carrier proteins, the titer of antibody induced may be further augmented with the use of appropriate immunological adjuvants. We have immunized groups of melanoma patients with vaccines either containing no adjuvant, or using DETOX, BCG or QS-21. QS-21 is a significantly more effective adjuvant than the others, producing significantly higher titer IgM and IgG antibodies. It is a saponin derivative extracted from the bark of the South American tree Quillaja saponaria Molina. The monosaccharide composition, molecular weight, adjuvant effect and toxicity for a series of these saponins have been described.^(35,36) It has also proven to be non-toxic and effective at augmenting the immunogenicity of an FeLV subunit vaccine in cats 37 and an HIV-1 recombinant vaccine in Rhesus monkeys. A phase I trial demonstrating the safety and suggesting the efficacy of a 100 ug QS-21 dose in patients treated with GM2-KLH vaccines has been reported. The only adverse events reported were minimal flu-like symptoms, and mild discomfort at the injection site.³⁸ Thus, conjugation with KLH and the addition of QS-21 have become standard approaches for vaccine construction at MSKCC has proven optimal for antibody induction against a variety of gangliosides, MUC1, MUC2, KSA, Tn, sTn, TF, Le^(y) and Globo H in the mouse and in humans.

3.6 Distribution of the antigens studied: In general, the antigens contained in this vaccine are expressed on ovarian cancer cells with high frequency. Recent studies at MSKCC have characterized this distribution in a variety of tumor types including ovarian cancer using immunohistochemical staining. A variety of tumor specimens were used in each tumor type, and it was required that 50% or more cells be positive in order to consider the antigen present. The presence of these antigens on the tumor specimens tested in ovarian cancer was: GM2 (100%), GLOBO-H (60%), MUC 1 (100%), sTn (60%), TF (100%), Le Y (80%).³⁹⁻⁴¹

3.7 Rational for the inventors' approach: The inventors' hypothesis is that induction of an antibody response against several cell surface antigens on ovarian cancer cells with a polyvalent conjugate vaccine will result in eradication of free tumor antigen, circulating tumor cells and micrometastases. The polyvalent nature of the vaccine and antibody response is important to eliminate escape by tumor cells that fail to express any one or two of the antigens, and to increase the number of antibodies reacting against each cell. It is expected that the inventors' vaccine will prove consistently immunogenic against five or six of the ovarian cancer cell surface antigens described above, and that it will prove nontoxic. Whether immunization with this polyvalent vaccine in high-risk ovarian cancer patients in the remission setting will result in prolonged disease-free and overall survival is the focus of subsequent studies to follow this pilot trial.

4.0 Vaccine Preparation

4.1 GM2 is provided by Progenics. GLOBO-H, Lewis-Y, TF(c), Tn (c), and sTn (c) are synthesized in the laboratory of Dr. Sam Danishefsky at the center. MUC-1 is produced at the core facility at MSKCC. For glycosylation, the MUC-1 was shipped to the University of Copenhagen, Glycobiology Group. The glycosylation was carried out by GalNAc transferase using UDP-N-GalNAc as substrate. The product was shipped back to MSKCC after purification by reverse phase HPLC.

4.2 QS-21 is obtained from Aquila Biopharmaceuticals in 100 mg vials as a white powder and is stored at −30 degrees Celsius. This is suspended initially in PBS as it is less soluble in normal saline and then final dilutions are made in normal saline. QS-21 is passed through a 0.22 micrometer filter immediately prior to use.

4.3 Conjugation to KLH is accomplished with three different conjugation procedures: direct amination (ozonolysis) for GM2, the M2C2H bifunctional linker group for Globo H and Le Y, and the MBS bifunctional linker group for the four mucin antigens.

4.4 The sterility of the conjugate is confirmed by passage through a 0.22 micrometer filter and it is stored in frozen normal saline at −30 to −80 degrees Celsius.

4.5 Vials are released for use following standard lot release testing (approximately five weeks).

5.0 Treatment Schedule and Dose

5.1 Vaccine contains:

[GM2(10 mcg)/TF(c)(3 mcg)/sTn(c) (3 mcg)/Globo-H(10 mcg)/MUC1-1-G5(3 mcg)/Le^(Y) (10 mcg)/Tn(c) (3 mcg)]-KLH (≈400 mcg) with adjuvant QS21(100 mcg)

5.2 Immunization Schedule

The vaccine will be administered at weekly intervals for 3 doses. This will be followed by a four week break and then a fourth vaccination. There will then be an eight week break and then a fifth vaccination, followed by additional immunizations every twelve weeks for 24 months total (as long as patient remains on study).

IMMUNIZATION SCHEDULE WEEK # 1 2 3 7 15 VACCINE # 1 2 3 4 5 5.3 Dose Administration and Modification

5.3.1 No dose escalation or dose modification will be performed. Systemic toxicity has not been seen with the previous vaccine studies. Systemic toxicity>grade II (with the exception of fever without infection) thought related to vaccination would result in removal of the patient from study and suspension of the protocol pending investigation.

5.3.2 The preparation will be administered subcutaneously to a site in the shoulders, buttocks, or thighs. It will be administered in one syringe, and will be supplied in approximately 1 cc total volume.

6.0 Evaluation During Study

6.1 Immune Function (Summarized in Table)

6.1.1 Antibody response: Peripheral blood (20-30 ml) will be drawn according to the schedule in table 8.3 with the exception of week 0, 7, and 9 at which time 50-60 ml will be collected for antibody testing. Thereafter, blood will be drawn at 12-week intervals as the patients return for booster immunizations. Antibodies against various antigens will be studies by ELISA, and against human tumor cell lines by FACS when appropriate.

6.2 Clinical and Laboratory Assessment:

6.2.1 Clinical and laboratory assessment schedule: The clinical and laboratory assessment schedule is outlined in Section 8.3 and includes parameters to assess the safety of the vaccine, as well as evaluate for signs of disease recurrence and progression. Abnormal findings will be evaluated per standard medical practice, and the abnormality will be classified as related to treatment, disease progression, or neither.

6.2.3 Extent of disease evaluation: All patients are by definition without clinical or radiographic evidence of disease at protocol entry.

6.2.4 Radiographic imaging (CT abdomen and pelvis) will be obtained q 6 months while on study, or if any clinical symptoms/examination findings warrant further evaluation, or if serum CA-125 rises to >70 (per time to treatment failure criteria), confirmed by repeat value; or at any time at the discretion of the investigator.

6.2.5 Length of follow-up: The primary endpoint of the study in this pilot trial is safety. Patients will be followed for the duration of the study, but based on previous trials, antibodies are generally present by the seventh week, and we will proceed with the proposal for additional studies to evaluate efficacy if no systemic toxicity is seen in any patient at the ninth week assessment. An additional B-12 weeks would be required for processing before patients could be enrolled on the polyvalent study, allowing ample time for follow-up of the pilot trial. Patients will be followed until time to treatment failure, or until all vaccinations are completed (maximum 24 months).

6.3 Summary of Evaluation:

SUMMARY OF CLINICAL, LABORATORY, AND IMMUNE ASSESSMENTS week # 0^(a) 1 2 3 5 7 9 13 15 17 27^(c) vaccine # 1 2 3 4 5 V Office Visit * * * * * * * * * * * Hx and PE * * * * * * * * * CA-125 * * * * * * CBC, diff * * * * * * * * * * Hepatic profile + * * * * * * * * * * creatinine Amylase * * * * * * * * * * Urinalysis * * * * * PT * Stool guaiac^(b) * * * * TSH * * Immune^(d) * * * * * * * bloods ^(a)= pretreatment evaluation, within 3 weeks ^(b)= stool guaiac may be obtained by digital exam or cards collected by patient ^(c)= following week 27, visits + laboratory studies + immunization q 3 months, CT scan q 6 months while on study ^(d)= immune bloods routinely consist of 20-30 ml collected in 3 red top tubes. In order to obtain sufficient serum to evaluate for multiple antibodies, 6 tubes will be collected instead of 3 at the pre-vaccination visit; and at week 7 and 9 only. 7.0 Biostatistical Considerations Endpoints:

The primary endpoints of this pilot trial are safety and confirmation of immunogenicity in the polyvalent setting. No systemic toxicity has occurred with the administration of monovalent vaccines at the center. Toxicity is not expected with this preparation. Following this pilot, additional studies with efficacy as the endpoint will be proposed. This pilot trial would be suspended pending investigation for any systemic toxicity thought related to vaccine in any patient. The same criteria for immunogenicity will be used as that of the individual pilot trials: patients must have IgM titer≧1:80, or a four fold increase in prevailing antibody titer if present at baseline. Nine patients will be accrued, and ≧5 of 9 patients should meet these criteria for three or more antigens in order to proceed with this construct in additional studies. In prior trials, antibodies are generally present by completion of the fourth vaccination, (week 7). If no systemic toxicity is seen by the week 9 assessment in these patients, we will proceed with proposals for phase II studies with efficacy endpoints.

While not the endpoint of this pilot, patients would be removed from study for relapse as defined below. Time to treatment failure will be simply defined based on data from Rustin et al.⁴² Treatment failure can be characterized by 1) physical examination evidence of disease recurrence, radiographic evidence of disease recurrence (biopsy will be performed at the discretion of the principal investigator but is not required); or 3) CA-125 elevation to twice the upper limits of normal (ie. ≧70), confirmed by a second sample also ≧70 U/ml. Time to treatment failure for biochemical relapse is recorded as the date of the first sample ≧70 U/ml.

The secondary endpoint of this pilot trial is to characterize the nature and duration of the immune response. Peripheral blood (20-30 ml) will be drawn as indicated in the table. Antibodies against the individual antigens will be studies by ELISA, and against the appropriate human tumor antigen FACS using human tumor cell lines expressing the respective antigen.

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Example 2 Heptavalent Vaccine Immunogenicity Trial in Mice

Methods

Serological Analyses

1. Elisa (Enzyme-Linked Immunosorbent Assay):

ELISA assays were performed as described below. Antigen in ethanol or in 0.1 M carbonate buffer (pH 11) were coated on ELISA plates at 0.2 μg/well for glycolipids and 0.1 μg/well for peptides. Serially diluted antiserum was added to each well and alkaline phosphatase-conjugated goat anti-mouse IgM or anti-mouse IgG was added at a dilution of 1:200 (Southern Biotechnology Associates, Inc, Birmingham, Ala.). Goat anti-mouse IgG and IgM conjugated with alkaline phosphatase obtained from Kierkegaard and Perry Labs, (Gaithersburg, Md.) were used as second antibodies. ELISA titer is defined as the highest dilution yielding an absorbance of 0.1 or greater over that of normal control mouse sera.

2. Cell Surface Reactivity Determined by FACS:

The cell surface reactivity of immune sera was tested on human MCF-7 and LSC cell lines. Single cell suspensions of 2×10⁵ cells/tube were washed in PBS with 3% fetal calf serum (FCS) and 0.01M NaN₃ and incubated with 20 μl of 1:20 diluted sera or monoclonal antibody mAb for 30 min on ice. After two washes with 3% FCS in PBS, 20 μl of 1:15 diluted goat anti-mouse IgM or IgG-labeled with fluorescein-isothiocyanate (FITC, Southern Biotechnology Associates Inc. Birmingham, Ala.) was added, and the mixture incubated for 30 min. After a final wash, the positive population and mean fluorescence intensity of stained cells were differentiated using FACScan, Becton & Dickinson Immunocytometry, San Jose, Calif.

Immunizaton of mice with Heptavalent-KLH Conjugates plus ELISA GPI100 Or QS21. Mar. 19, 2001 ELISA mice Immunized with . . . / pre vacc. post vacc. plate coated with . . . (Group#) (IgG/IgM) (IgG/IgM) Comments GM2 GM2 (group #1) 0/0 0/0 GloboH (group#2) 0/0 LeY (group #3) 0/0 Muc1-G5 (group #4) 0/0 STn(c) (group #5) 0/0 TF(c) (group #6) 0/0 Tn(c) (group #7) 0/0 Globo H GM2 (group #1) GloboH (group#2) 0/0  640/5120 LeY (group #3) Muc1-G5 (group #4) STn(c) (group #5) TF(c) (group #6) Tn(c) (group #7) LeY GM2 (group #1) 0/0 GloboH (group#2) 0/0 LeY (group #3) 0/0  640/2560 Muc1-G5 (group #4) 0/0 STn(c) (group #5)  0/160 TF(c) (group #6)  0/40 Tn(c) (group #7)  0/40 Muc1-G5 GM2 (group #1)  0/320 GloboH (group#2)  0/160 LeY (group #3)  0/160 Muc1-G5 (group #4) 0/0 2560+/1280  STn(c) (group #5) 40/80 TF(c) (group #6) 40/80 Tn(c) (group #7)  0/320 mAb. C595 2560/ B55 /<5120+++ STn(c) GM2 (group #1) 0/0 GloboH (group#2) 0/0 LeY (group #3) 0/0 Muc1-G5 (group #4) 0/0 STn(c) (group #5) 0/0 5120/320  TF(c) (group #6) 0/0 Tn(c) (group #7) 0/0 mAb. cc49 1280/0   Tf(c) GM2 (group #1)  0/40 GloboH (group#2) 40/80 LeY (group #3) 40/40 Muc1-G5 (group #4) 5120+/160  STn(c) (group #5) 5120+/320  TF(c) (group #6) 0/0 5120+/320  Tn(c) (group #7) 5120+/640  mAb. JAA-F11 0/ A78-6/A7 /640 Tn(c) GM2 (group #1) 0/0 GloboH (group#2) 0/0 LeY (group #3) 0/0 Muc1-G5 (group #4) 5120+/160  STn(c) (group #5) 5120+/320  TF(c) (group #6) 5120+/320  Tn(c) (group #7) 0/0 5120+/2580  mAb. 5F4 /5120++ HB-Tn1 /5120

Immunization of mice with Heptavalent-KLH Conjugates* plus GPI-100 or QS-21. ELISA (IgG/IgM) FACS (IgG/IgM) Group # Antigen Mice # Pre-vacc. Post-vacc Pre-vacc. Post-vacc. Comments 8. Heptavalent-KLH + Tn(c) 1 0/0 5120+/160  100 ug GPI-100 2 0/0 5120/160  (200ul/mouse) 3 0/0 5120/160  4 0/0 5120++/640   5  0/40 5120+/320  Tf(c) 1 0/0 5120/160  2 0/0 5120/160  3 0/0 5120/160  4 0/0 5120+/320  5  0/40 5120+/640  sTN(c) 1 0/0 5120/640  2 0/0 1280/640  3 0/0 5120/640  4 0/0 5120/320  5 0/0 1280/320  MUC1-1G5 1 0/0 5120+/0   2 0/0 5120+/80    3 0/0 5120+++/160    4 0/0 5120++/40   5 0/0 5120+++/80     Ley 1 0/0  0/40 2 0/0 320/640 3 0/0  0/160 4 0/0 80/40 5 0/0  0/640 Globo H 1 0/80  40/640 2  0/80  0/640 3  0/80  0/640 4  0/160  40/1280 5  0/160   0/2560 GM2 1 0/0  0/40 2 0/0 0/0 3 0/0 0/0 4 0/0 0/0 5 0/0 0/0 Heptavalent: Tn(c) (3 ug), TF(c) (3 ug), sTn(c) (3 ug), MUC1-1G5 (3 ug), Ley (10 ug), Globo H (10 ug), and GM2 (10ug). New GPI-100 used for all groups except group 9 (batch J). Polysorbate 80, 4 mg/ml in final vaccine.

Immunization of mice with Heptavalent-KLH Conjugates* plus GPI-100 or QS-21. ELISA (IgG/IgM) FACS (IgG/IgM) Group # Antigen Mice # Pre-vacc. Post-vacc Pre-vacc. Post-vacc. Comments Apr. 2, 2001 9. Heptavalent-KLH + Tn(c) 1 0/0 640/40  100ug GPI-100 2 0/0 640/0  (Batch J) 3 0/0 5120/40  (200 ul/mouse) 4 0/0 5120+/0   5 0/0 1280/40  Tf(c) 1 0/0 1280/80  2 0/0 1280/40  3 0/0 5120+/40   4 0/0 5120++/40   5 0/0 2560/40  sTN(c) 1 0/0 320/80  2 0/0 320/160 3 0/0 640/160 4 0/0 640/80  5 0/0  80/320 MUC1-1G5 1 0/0 2560/0   2 0/0 640/0  3 0/0 640/0  4 0/0 640/0  5 0/0 2560/80  Ley 1 0/0 0/0 2 0/0  0/80 3 0/0 0/0 4 0/0 0/0 5 0/0  160/1280 Globo H 1  0/80  0/160 2  0/80  0/320 3  0/80  0/160 4  0/80  0/320 5  0/80  40/320 GM2 1 0/0 0/0 2 0/0 0/0 3 0/0 0/0 4 0/0 0/0 5 0/0 0/0 10. Heptavalent-KLH + Tn(c) 1 0/0 5120++/320   100 ug GPI-100 + 2 0/0 5120/320  polysorbate 80 3  0/40 5120+/1280  (200 ul/mouse) 4 0/0 5120+/640  5 0/0 2560/320  Tf(c) 1 0/0 5120+/320  2 0/0 5120+/160  3 0/0 5120+/640  4 0/0 5120+/640  5 0/0 5120+/320  sTN(c) 1 0/0 320/640 2 0/0  320/1280 3 0/0 1280/1280 4 0/0 640/40  5 0/0 320/320 MUC1-1G5 1 0/0 1280/40  2 0/0 5120/80  3 0/0 5120/160  4 0/0 2560/160  5 0/0 5120/40  Ley 1 0/0 0//0 2 0/0 0/0 3 0/0  0/640 4 0/0  0/640 5 0/0  0/80 Globo H 1  0/40  80/640 2  0/80  40/2560 3  0/320   0/2560 4  0/160   0/2560 5  0/80  0/640 GM2 1 0/0 0/0 2 0/0 0/0 3 0/0 0/0 4 0/0 0/0 5 0/0 0/0 11. Heptavalent-KLH ++ Tn(c) 1 0/0 5120/160  10 ug QS-21 2 0/0 5120+/160  (200 ul/mouse) 3 0/40 5120+++/160    4  0/80 2560/320  5 0/0 5120+/80   Tf(c) 1  0/40 5120+/320  2 0/0 5120++/640   3  0/160 5120++640 4  0/320 2560/640  5  0/40 5120++/320   sTN(c) 1 0/0 40/40 2 0/0 2560/640  3 0/0 5120/160  4 0/0 1280/80  5 0/0 5120/640  MUC1-1G5 1 0/0 1280/160  2 0/0 2560/80  3 0/0 5120/80  4 0/0 2560/320  5 0/0 5120++/80   Ley 1 0/0 0//0 2 0/0 0/0 3 0/0 0/0 4 0/0 0/0 5 0/0 40/40 Globo H 1  0/80  40/320 2  0/80  80/320 3  0/80  40/640 4  40/160  0/640 5  0/320   0/2560 GM2 1 0/0 0/0 2 0/0 0/0 3 0/0 0/0 4 0/0 0/0 5 0/0 0/0 Apr. 16, 2001 12. Heptavalent-KLH + Tn(c) 1 0/40 2560/160 3 ug ER-803022 2 0/0 640/160 (200 ul/mouse) 3  0/40 320/320 4 0/0 160/160 5 0/0 2560/160  Tf(c) 1 0/0 2560/40  2 0/0 2560/40  3 0/0 640/160 4 0/0 320/80  5 0/0 2560/0   sTN(c) 1 0/0  0/40 2 0/0 40/80 3 40/40  80/160 4 0/0 160/160 5 0/0 40/0  MUC1-1G5 1 0/0 80/0  2 0/0 80/0  3 0/0 80/40 4 0/0 40/80 5 0/0 40/0  Ley 1 0/0 0/0 2 0/0 0/0 3 0/0 0/0 4 0/0  0/80 5 0/0 0/0 Globo H 1  0/80  0/320 2  0/160  40/1280 3  0/320  0/320 4  80/160  160/1280 5  40/160 320/640 GM2 1 0/0 0/0 2 0/0 0/0 3 0/0 0/0 4 0/0 0/0 5 0/0 0/0 13. Heptavalent-KLH + Tn(c) 1  0/40  40/320 10 ug ER-803732 2 0/0 40/40 (200 ul/mouse) 3 0/0 320/160 4  0/40 160/160 5  0/40 160/80 Tf(c) 1 0/0 160/160 2 0/0 320/40  3  0/40 640/160 4  0/40 320/160 5 0/0 320/160 sTN(c) 1 0/0 40/80 2 0/0  0/80 3 0/0  0/320 4  0/160  40/320 5  0/40  40/160 MUC1-1G5 1 0/0 640/40  2 0/0  0/40 3 0/0 40/80 4  0/40  80/160 5 0/0 160/80  Ley 1 0/0 0/0 2 0/0 0/0 3  0/40  40/320 4 0/0  0/40 5 0/0  0/80 Globo H 1 40/80  80/160 2  0/160  40/320 3  0/40  80/320 4  0/80  0/160 5  80/160 160/320 GM2 1 0/0 0/0 2 0/0 0/0 3 0/0 0/0 4 0/0 0/0 5 0/0 0/0 14. 30 ug KLH + Tn(c) 1 0/0  0/160 100 ug GPI-100 2 0/0  0/40 (100 ul/mouse) 3 0/0 0/0 4 0/0  0/160 5  0/40  0/640 Tf(c) 1  0/40  0/320 2  0/40  0/160 3 0/0  0/80 4 0/0  0/320 5  0/160  40/640 sTN(c) 1 0/0  0/80 2 0/0  0/40 3 0/0 0/0 4 0/0  0/320 5 0/0  0/80 MUC1-1G5 1 40/40  0/160 2  0/40  0/80 3 0/0  0/80 4 0/0  0/320 5 0/0  0/320 Ley 1 0/0  0/40 2 0/0 0/0 3 0/0 0/0 4 0/0 0/0 5 0/0 0/0 Globo H 1  0/320  40/1280 2  0/320  0/640 3  0/80  0/1280 4  0/160  0/640 5  0/160  40/640 GM2 1 0/0  0/160 2 0/0  0/40 3 0/0 0/0 4 0/0  0/320 5 0/0  0/160 KLH 1 0/0 5120++/320   2  0/40 5120++/160   3 0/0 5120++/320   4 0/0 5120++/640   5  0/40 5120++/320   FACS % positive cells Group # MCF-7 (IgG/IgM) LSC (IgG/IgM) (mice immunized with . . .) Mice # Pre-vacc. Post-vacc. 1:200 dilut. Pre-vacc. Post-vacc. 1:200 dilut. 1. 3 ug GM2-KLH + 1 11.79/10.68 21.45/11.79 15.73/0.38  10.90/9.58  6.91/2.93 6.56/0.14 100 ug GPI-100 2 10.23/10.69 4.80/1.49 2.34/0.0  10.90/10.45 5.99/3.45 1.45/0.59 (100 ul/mice) 3  9.50/10.74  6.95/15.79 0.99/0.21 11.29/11.11 7.79/1.53 6.41/0.23 4 9.81/9.68  3.41/12.63 0.99/2.06  9.76/11.06 6.94/1.75 3.42/0.02 5 10.81/10.42 10.43/3.15  12.29/0.11  10.13/10.76 4.40/1.67 0.40/0.10 2. 3 ug GloboH-KLH + 1 10.63/9.93   5.79/17.26 6.60/0.19  9.87/10.99 7.08/2.77 9.55/0.25 100 ug GPI100 2 10.08/10.67 16.60/27.63 10.54/6.75  10.0/10.41  8.9/3.43 1.29/0.52 (100 ul/mice) 3 11.03/10.09 27.21/40.86 27.58/2.30  10.42/10.61 11.56/1.15  7.60/0.02 4 10.16/11.36 1.92/13.77 2.30/1.03 10.57/11.04 5.29/2.43 3.81/0.24 5 11.94/10.82 24.61/12.97 20.32/3.62  10.79/10.72 10.58/2.54  1.25/0.20 3. 3 ug Ley-KLH + 1 11.01/9.97  85.01/43.57 41.26/1.73  10.22/10.10 89.17/44.27 13.40/1.87  100 ug GPI-100 2 9.95/9.69 47.75/78.17 4.86/3.43 10.49/8.39  91.69/79.9  7.64/7.04 (100 ul/mice) 3 10.41/10.58 69.55/10.95 20.53/0.12  11.30/9.47  95.57/21.25 24.67/0.16  4 11.93/10.03 91.08/0.76  21.29/0.0 10.96/9.60  81.5/6.24 9.08/0.38 5 12.46/11.18 42.94/36.67 13.66/1.87  10.98/10.61 12.74/52.16 1.05/1.87 4. 3 ug Muc-1G-5-KLH + 1 11.33/10.94 98.12/9.52  97.98/0.93  9.50/9.89 4.83/1.10 2.29/0.18 100 ug GPI-100 2 10.90/9.44 88.05/1.47  66.29/0.07  11.39/9.63  8.47/0.15 4.85/0   (100 ul/mice) 3 10.59/10.81 93.34/0.93  83.12/0.09  10.42/9.85  7.42/1.16 3.15/0.02 4 10.47/9.70  94.57/21.92 85.52/0.47   9.70/11.11 10.99/7.79  3.47/0.71 5 12.29/10.03 99.77/6.12  99.79/0.77  *11.05/10.49  63.71/1.82  28.90/0.71  5. 3 ug STn(c)-KLH + 1 9.56/9.77 13.95/1.74  1.29/0.13 10.10/10.73 95.14/4.42  86.94/0.50  100 ug GPI-100 2  9.91/10.59 15.95/1.79  12.70/0.23  10.20/11.18 98.65/19.42 88.58/0.59  (100 ul/mice) 3 10.72/10.55 6.88/2.81 6.23/0.02 11.31/11.48 97.78/31.98 95.31/0.36  4 9.62/9.52 4.62/1.69 3.18/0.14 10.11/9.78  94.14/1.19  88.65/0.09  5 10.77/10.71 17.96/2.16  7.17/0.3  10.71/0.05  97.66/7.28  87.09/0.38  6. 3 ug Tf(c)-KLH + 1 10.84/10.39 13.89/5.73  5.86/0.23  9.64/10.31 7.66/4.74 3.85/0.24 100 ug GPI-100 2  9.22/11.34 31.03/4.44  31.75/0.10   9.65/10.15 9.01/1.17 3.98/0.18 (100 ul/mice) 3 10.59/10.11 4.05/1.05 0.73/0.02 10.12/9.92  4.97/2.77 0.90/0.04 4 10.26/10.63 6.72/7.10 2.19/0.19  9.72/10.79 3.85/3.58 2.25/0.16 5  12.0/10.90 9.11/6.28 5.18/0.18 *11.54/10.09  11.71/5.0  12.76/0.64  7. 3 ug Tn(c)-KLH + 1  9.60/10.44 3.64/8.41 0.62/0.37 10.65/10.46 9.91/5.07 3.02/0.32 100 ug GPI-100 2 10.54/10.46 12.94/3.24 20.35/0.17  11.50/10.85 4.81/0.48 5.33/0.05 (100 ul/mice) 3 10.51/10.46  4.72/23.22 0.16/1.22 10.70/9.68  2.37/1.33 0.04/0.02 4 10.97/9.48   8.50/17.77 4.52/0.16 10.94/10.97 18.50/1.81  4.18/0.12 5 10.79/10.72 46.27/32.77 9.46/2.04 11.40/10.16 25.42/16.84  16.5/22.80 * DU145 +control IE-3 91.67, 95.15 +control IE-3 95.73% MLS128 77.07% HMFG1 38.34% MLS132 19.92, 4.52 49H8 84.90% 8. Heptavalent-KLH + 1 10.84/11.21 89.44/17.63 69.48/0.47  10.29/10.63 97.79/49.78 90.80/0.46  100 ug GPI-100 2 9.55/9.54 96.98/25.87 83.67/1.12  9.97/10.58 95.44/74.27 83.11/0.35  (200 ul/mice) 3  9.94/10.88 96.53/32.44 88.36/0.79  10.56/10.91 95.39/40.29 85.79/0.49  4 10.90/10.72 99.47/5.72  96.61/0.45  10.69/10.42 97.65/16.44 88.77/0.28  5 10.37/10.17 75.64/10.67 63.57/0.11  10.58/10.14 93.93/15.49 71.06/1.36  9. Heptavalent-KLH + 1 10.65/10.57 74.25/12.81 69.56/1.07  10.93/10.21 99.45/8.83  85.59/0.38  100 ug GPI-100 2 10.20/10.97 98.22/16.51 81.61/0.91  10.86/10.78 96.70/22.94 66.20/0.50  (batch J) 3 9.96/9.68 89.36/1.03  60.88/0.22  9.79/9.80 95.10/0.43  53.45/0.03  (200 ul/mice) 4 10.77/10.75 82.25/8.95  27.80/0.09  10.58/10.28 92.21/1.17  65.13/0.18  5 10.95/10.77 77.49/10.19 62.39/0.13  10. Heptavalent-KLH + 1 10.99/10.32 84.54/8.61  39.91/0.49  10.63/10.92 93.11/38.33 63.61/1.57  100 ug GPI-100 + 2 10.09/10.92 97.83/11.79 94.21/0.16  10.72/10.68 97.64/12.52 77.95/0.05  polysorbate 80 3 11.32/10.32 97.74/15.67 89.24/0.30   9.53/10.16 89.84/2.13  63.96/0.19  (200 ul/mice) 4  9.76/10.59 92.51/15.57 82.83/0.10  11.50/10.03 60.13/14.56 8.56/1.59 5 10.33/10.05 80.12/11.34 70.98/0.30  11. Heptavalent-KLH + 1 10.96/10.08 91.06/1.64  55.98/0.02   9.76/10.81 26.58/2.13  5.42/0.16 10 ug QS-21 2  9.88/10.41 94.84/2.32  72.07/0.02   9.80/11.58 88.16/22.37 34.36/0.94  (200 ul/mice) 3  11.0/10.57 99.34/9.99  93.96/0.84   9.86/10.79 87.20/11.70 76.90/0.14  4 11.05/10.65 88.58/3.78  61.69/0.13  10.07/10.45 71.56/3.79  26.42/0.15  5 10.86/10.27 81.44/6.27  69.68/0.14  12. Heptavalent-KLH + 1 18.54/11.29 67.73/1.59  26.33/0.18  10.98/9.97  16.98/1.97  6.40/0.09 3 ug ER803022 2 9.69/9.60 74.76/0.72  33.45/0.08  10.62/10.33 36.76/2.05  3.73/0.07 (200 ul/mice) 3  9.65/10.62 91.62/2.89  46.96/0.31  10.16/10.03 28.33/4.88   3.26/0.10 4 10.78/10.0  22.58/4.14  9.51/0.36 5 10.90/10.94 48.15/7.29  10.64/0.23  13. Heptavalent-KLH + 1 10.08/10.56 74.85/8.71  40.57/0.24  10.96/10.20 64.39/3.85  7.58/0.17 10 ug ER803732 2 11.03/10.91 36.35/5.07  4.79/0.07  9.84/10.19  3.67/11.73 0.47//0.73 (200 ul/mice) 3 10.22/10.54 65.94/30.22 7.87/0.63 10.83/10.07 66.06/25.19 8.66/0.82 4 10.75/10.07 91.69/12.15 22.96/0.41  5 10.87/11.04 35.45/6.88  1.38/0.11 14. 30 ug KLH + 1  9.42/11.35 13.72/9.84  10.08/2.29  10.85/10.49 5.49/4.48 3.29/2.53 100 ug GPI-100 2 12.14/11.31 17.50/2.09  6.41/0.0  10.58/10.44 10.57/3.53  14.38/0.21  (200 ul/mice) 3  9.82/10.97 1.64/1.05 0.13/0.02  9.19/11.45 15.10/1.03  11.63/0.01  4 11.20/11.84 13.25/2.14  10.05/0.12  5 10.51/9.95  8.98/5.40 1.38/0.06 Heptavalent: Tn(c) (3 ug), TF(c) (3 ug), sTn(c) (3 ug), MUC1-1G5 (3 ug), Ley (10 ug), Globo H (10 ug), and GM2 (10 ug). New GPI-100 used for all groups except group 9 (batch J). Third Series of Experiments Polyvalent Conjugate Vaccine for Cancer

Tumor-specific antigens have been identified and pursued as targets for vaccines. The inventors' previous work has shown that monovalent vaccines utilizing the tumor antigens Globo H, Lewisy, GM2, glycosylated MUC-1, Tn(c), sTn(c), or TF(c) conjugated to KLH to be safe with local erythema and edema but minimal systemic toxicities. As a result of vaccination with these monovalent vaccines, most patients generated specific high titer IgM or IgG antibodies against the respective antigen-KLH conjugates. The present invention provides multivalent vaccines wherein the components of the monovalent vaccine are combined and administered with an adjuvant as treatment for cancer.

Vaccines consisting of a combination of tumor antigens administered with a saponin immunological adjuvant QS-21 or GPI-0100. The antigens are glycosylated MUC-1-G5, Globo H, GM2, Le^(y), Tn(c), sTn(c), and TF(c). In each case the antigen is conjugated to Keyhole Limpet Hemocyanin (KLH).

The preferred ranges of the antigen and adjuvant doses are as follows:

-   -   Glycosylated MUC-1-G5: 0.1 to 30 g;     -   Globo H: 0.1 to 100 g;     -   GM2: 0.1 to 100 g;     -   Le^(y): 0.1 to 60 g;     -   Tn(c): 0.1 to 100 g;     -   sTn(c): 0.1 to 100 g;     -   TF(c): 0.1 to 30 g;     -   QS-21: 25-200 g;     -   GPI-0100: 1-25 mg.

Example 1 Comparison of the Immune Response after Immunization with Monovalent and Hexavalent-KLH Conjugate Vaccines Against Prostate Cancer

The immune response of the five initial patients receiving hexavalent vaccine with the immune responses of patients who had previously been immunized with the respective monovalent vaccines is compared in the following five tables. Shown are the reciprocal mean peak ELISA titer for IgM and IgG after immunization and FACS assay (% of positive cells/mean intensity) using the MCF-7 tumor cell line. The comparison for GM2-KLH is pending. Comparing the responses induced by monovalent and hexavalent vaccines, there was no significant difference in the antibody responses against any of the five antigens tested to date. Combination of six individual conjugates into a single vaccine does not significantly change the antibody response against the individual antigens.

Hexavalent Versus Monovalent: Prostate Aug. 6, 2001 Patient Elisa (TF-HSA) MCF-7 FACS Trial Patient Sera IgM IgG IgM %/Mean protocol 98-048 M1 week 1  10 10 10/37 TF(c)-KLH + QS21 week 7  1280+ 160 11/39 dosage: 1 μg week 9 1280 40 17/50 M2 week 1  10 0 10/21 week 7  1280++ 10 52/54 week 9   1280+++ 10 72/64 M3 week 1   0 0  11/135 week 7  1280++ 160  2/28 week 9  1280++ 1280  6/104 M4 week 1   0 0 10/32 week 7  160 10 18/43 week 9  160 160 26/47 M5 week 1   0 0 10/36 week 7  320 0  9/28 week 9  320 0  8/22 protocol 00-064 H1 week 1  20 0 11/64 Hexavalent Conjgate + QS21 week 7 1280 160  7/55 TF(c)dosage: 3 μg week 12 1280 160  3/31 H2 week 1  40 10  9/37 week 7 1280 160 21/50 week 12  640 160 17/43 H3 week 1  40 0 10/35 week 7 1280 20 54/65 week 12  640 40 34/47 H4 week 1  80 0 10/26 week 7 1280 80 22/43 week 12 1280 20 19/54 H5 week 1   0 0 10/13 week 7 1280 320 57/26 week 12 1280 320 47/22 Controls C1 Aug. 27, 1999 1280 1280 Aug. 10, 2001 Patient Elisa (Tn-HSA) MCF-7 FACS Trial Patient Sera IgM IgG IgM %/Mean protocol 98-002 M1 week 1  0 80 10/22 Tn(c)-KLH + QS21 week 7  40 2560  7/18 dosage: 3 μg week 9  80 2560  9/21 M2 week 1  10 80 11/26 week 7 320 5120 32/40 week 9 640 5120 37/42 M3 week 1  20 40 11/25 week 7 640 640 12/26 week 9 1280  1280 13/27 M4 week 1  40 40 10/49 week 7 640 1280  9/40 week 9 1280  1280  8/39 M5 week 1  10 80 11/25 week 7 1280  5120 12/26 week 9 1280  5120 13/27 protocol 00-064 H1 week 1  80 0 11/64 Hexavalent Conjgate + QS21 week 7 320 1280 7/55 Tn dosage: 3 μg week 12 320 1280  3/31 H2 week 1 320 10  9/37 week 7 1280  320 21/50 week 12 1280  320 17/43 H3 week 1  10 0 10/35 week 7 320 640 54/65 week 12 640 640 34/47 H4 week 1  40 160 10/26 week 7 640 5120 22/43 week 12 320 5120 19/54 H5 week 1  0 20 10/13 week 7 320 640 57/26 week 12 320 640 47/22 Controls C2 1280+ 640 Aug. 17, 2001 Patient Elisa (GloboH) MCF-7 FACS Trial Patient Sera IgM IgG IgM %/Mean PROTOCOL 96-055 M1 week 1 0 0 11/40 GloboH-KLH + QS21 week 7 20 0  9/30 dosage: 10 μg week 9 20 0  9/40 M2 week 1 0 0 11/33 week 7 0 0 12/42 week 9 0 0 14/52 M3 week 1 20 0 week 7 640 0 week 9 640 0 M4 week 1 10 0 11/26 week 7 160 0 19/39 week 9 160 0 25/39 M5 week 1 40 0 10/26 week 7 160 0 17/38 week 9 off study off study PROTOCOL 00-064 H1 week 1 40 0 11/41 Hexavalent Conjgate + QS21 week 7 160 0  9/36 GloboH dosage: 10 μg week 12 160 80  6/27 H2 week 1 160 0 11/33 week 7 640 0 11/36 week 12 320 0 15/40 H3 week 1 40 0 11/29 week 7 20 0 56/59 week 12 20 0 43/46 H4 week 1 80 0 10/34 week 7 160 0 21/46 week 12 80 0 16/44 H5 week 1 40 0 10/15 week 7 80 0 64/35 week 12 80 0 45/27 Controls C3 week 26 640 C4 1280 Aug. 31, 2001 Patient Elisa Ley-Cer MCF-7 FACS Trial Patient Sera IgM IgG IgM %/Mean PROTOCOL 00-075 M1 week 1 0 0 11/23 LewY-MMCCH-KLH week 7 0 0 13/26 dosage: 20 μg week 9 0 0 24/92 M2 week 1 0 0 10/25 week 7 0 0 20/32 week 9 0 0 14/24 M3 week 1 0 0 11/30 week 7 40 80  8/20 week 9 20 40 14/41 M4 week 1 0 0 11/46 week 7 0 0 10/50 week 9 0 10  9/44 M5 week 1 0 0 10/50 week 7 40 0  3/30 week 9 0 10 18/63 PROTOCOL 00-064 H1 week 1 0 0 11/41 Hexavalent Conjgate + QS21 week 7 0 0  9/36 Ley dosage: 10 μg week 12 0 0  6/27 H2 week 1 0 0 11/33 week 7 0 0 11/36 week 12 0 0 15/40 H3 week 1 0 0 11/29 week 7 10 0 56/59 week 12 0 0 43/46 H4 week 1 0 10 10/34 week 7 10 10 21/46 week 12 10 0 16/44 H5 week 1 0 0 10/15 week 7 0 0 64/35 week 12 0 0 45/27 Controls C5 2560 C6 640 Aug. 19, 2001 Patient Elisa (MUC33G5) MCF-7 FACS Trial Patient Sera IgM IgG IgM %/Mean PROTOCOL 99-23 M1 week 1 0 0 10/25 MUC33G 5 site-KLH + QS21 week 7 160 640 17/31 dosage: 3 μg week 9 160 640 31/45 M2 week 1 0 0 10/29 week 7 2560 640 38/53 week 9 2560 640 35/43 M3 week 1 0 20 10/28 week 7 2560 160 36/57 week 9 2560 320 43/60 M4 week 1 0 0 11/41 week 7 2560 80 12/35 week 9 640 320 12/38 M5 week 1 0 0 11/30 week 7 40 0 61/65 week 9 40 80 59/67 PROTOCOL 00-064 H1 week 1 0 0 11/41 Hexavalent Conjgate + QS21 week 7 20 640  9/36 MUC33 dosage: 3 μg week 12 0 160  6/27 H2 week 1 0 0 11/33 week 7 0 40 11/36 week 12 0 80 15/40 H3 week 1 0 0 11/29 week 7 160 160 56/59 week 12 40 320 43/46 H4 week 1 0 20 10/34 week 7 40 320 21/46 week 12 40 160 16/44 H5 week 1 0 0 10/15 week 7 40 160 64/35 week 12 0 80 45/27 Controls C7 2560 640

Immunization of mice with Heptavalent-KLH Conjugates* plus GPI-100 or QS-21 Mean Value: Nov. 30, 2001 LSC ELISA FACS MCF-7 # LSC Pre Post Pre Post 1:200 Pre Post 1:200 CDC (MCF-7) Group # Antigen IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM Mice # Pre Post 1. 3 ug Tn 10.43% 10.44% 9.40% 8.97% 6.47% 0.55% 10.59% 10.60% 6.41% 1.92% 3.63% 0.22% GM2-KLH + Tf 100 ug GPI-100 sTn (100 ul/mice) Muc1-G5 Ley globo H GM2 0 0   0 0 2. 3 ug Tn 10.77% 10.57% 15.22% 22.50% 13.47% 2.78% 10.33% 10.75% 7.08% 2.46% 4.70% 0.25% GloboH-KLH + Tf 10.08% 10.57% 8.01% 32.59% 6.86% 7.39% 100 ug GPI100 sTn (mice#1 (100 ul/mice) re-tested.) Muc1-G5 Ley globo H 0 0  640 5120 GM2 3. 3 ug Ley-KLH + Tn 11.15% 10.34% 67.27% 34.02% 20.32% 1.43% 10.79% 9.63% 74.13% 40.76% 11.12% 2.26% 3-1 5.94% 46.42%  100 ug GPI-100 Tf 3-2 4.47% 75.05%  (100 ul/mice) sTn 3-3 4.49% 9.21% Muc1-G5 3-4 22.5% 119.0%  Ley 0 0  640 2560 3-5 11.02%  61.7% globo H GM2 4. 3 ug Tn 11.12% 10.12% 94.77% 7.99% 86.54% 0.46% 10.25% 10.12% 7.93% 2.55% 3.44% 0.23% 4-1 3.30% Muc-1G-5-KLH + Tf 4-2 6.85% 100 ug GPI-100 sTn 4-3 14.0% (100 ul/mice) Muc1-G5 0 0   2560+ 1280 4-4 4.49% Ley 4-5 4.23% globo H GM2 5. 3 ug Tn 10.12% 10.23% 11.88% 2.04% 6.11% 0.16% 10.49% 8.64% 96.67% 12.86% 88.91% 0.38% 5-1 6.83% STn(c)-KLH + Tf 5-2 10.91%  100 ug GPI-100 sTn 0 0 5120 320 5-3 6.36% (100 ul/mice) Muc1-G5 5-4 7.11% Ley 5-5 4.37% globo H GM2 6. 3 ug Tf(c)-KLH + Tn 10.58% 10.67% 10.56% 4.92% 9.14% 0.14% 9.78% 10.29% 6.37% 3.06% 2.74% 0.16% 100 ug GPI-100 Tf 0 0 5120 320 (100 ul/mice) sTn Muc1- G5 Ley globo H GM2 7. 3 ug Tn 0 0   5120+ 2560 10.48% 10.31% 15.21% 17.10% 7.02% 0.79% 11.04% 10.42% 12.20% 5.11% 5.81% 4.66% Tn(c)-KLH + Tf 100 ug GPI-100 sTn (100 ul/mice) Muc1-G5 Ley globo H GM2 KLH 8. Heptavalent- Tn 0 10   5120+ 320 10.32%  10.5% 91.61% 18.46% 80.34% 0.59% 10.43% 10.54% 96.04% 39.25% 83.91% 0.59% 8-1 0.75% 2.99% KLH + Tf 0 10 5120 320 10.63% 10.14% 98.92% 24.41% 85.27% 7.44% 8-2  1.0% 12.63%  100 ug GPI100 sTn 0 0 2560 640 (mice #1 8-3 −2.23%   44.48%  (200 ul/mice) re-tested) Muc1-G5 0 0     5120++ 80 8-4 −3.12%   24.77%  Ley 0 0  80 160 8-5 7.65% 27.28%  globo H 0 80  10 1280 VK9 2.05% GM2 0 0   0 10 3S193 82.2% anti-GM2 71.2% 696 32.2% 9. Heptavalent- Tn 0 0 2560 40 10.51% 10.55% 84.31% 9.89% 60.45% 0.48% 10.40% 10.21% 92.22% 16.68% 63.24% 0.25% 9-1 −2.79%   34.02%  KLH + Tf 0 0 5120 40 9-2 2.96% 9.42% 100 ug GPI (old sTn 0 0  320 160 9-3 4.50% 11.41%  batch) Muc1-G5 0 0 1280 0 9-4 6.74% 9.51% (200 ul/mice) Ley 0 0  40 320 9-5 4.01% 39.91%  globo H 0 80   0 320 3S193 97.83%  GM2 0 0   0 0 B72.3 5.34% HMFG.1 3.01% 10. Heptavalent- Tn 0 0   5120+ 640 10.50% 10.44% 90.55% 12.59% 75.43% 0.27% 10.67% 10.24% 85.22% 11.17% 54.16% 0.74% 10-1 −6.29%   −3.3% KLH + Tf 0 0 5120 320 100 ug GPI-100 + sTn 0 0  640 640 polysorbate 80 Muc1-G5 0 0 5120 80 (200 ul/mice) Ley 0 0   0 320 globo H 0 160  20 1280 GM2 0 0   0 0 11. Heptavalent- Tn 0 20   5120+ 160 10.75% 10.39% 90.96% 4.8% 70.67% 0.23% 9.92% 10.74% 70.28% 8.33% 40.71% 0.29% 11.1 1.84% 10.93%  KLH + Tf 0 80   5120+ 640 11-2 0.71% 3.20% 10 ug QS-21 sTn 0 0 2560 320 11-3 1.31% 5.88% (200 ul/mice) Muc1-G5 0 0 2560 160 11-4 −1.58%   18.36%  Ley 0 0  10 10 11-5 10.28%  27.09%  globo H 0 80  40 640 GM2 0 0   0 0 12. Heptavalent- Tn 0 0 1280 160 11.89% 10.49% 60.97% 3.33% 25.38% 0.23% 10.42% 10.29% 29.79% 4.55% 5.65% 0.14% KLH + Tf 0 0 1280 80 3 ug ER803022 sTn 0 0  80 80 (200 ul/mice) Muc1-G5 0 0  80 20 Ley 0 0   0 20 globo H 20 160  80 640 GM2 0 0   0 0 13. Heptavalent- Tn 0 20  160 160 10.59% 10.62% 60.86% 12.60% 15.51% 0.29% 10.50% 10.24% 45.30% 11.86% 5.60% 0.60% KLH + Tf 0 10  320 160 10 ug ER803732 sTn 0 40  20 160 (200 ul/mice) Muc1-G5 0 0  160 80 Ley 0 0   0 80 globo H 20 80  80 320 GM2 0 0   0 0 14. 30ug KLH + Tn 0 0   0 160 10.62% 11.08% 11.02% 4.10% 5.61% 0.45% 10.32% 10.68% 9.09% 2.47% 7.96% 0.73% 100 ug GPI-100 Tf 0 40   0 320 (100 ul/mice) sTn 0 0   0 80 Muc1-G5 0 10   0 160 Ley 0 0   0 0 globo H 0 160  10 640 GM2 0 0   0 160 KLH 0 10     5120++ 320

Immunization of mice with Heptavalent-KLH Conjugates* plus QS-21 or GPI 100 with or without mAb 9H10 against CTLA-4. Mean Value: ELISA (mean) ELISA (median) FACS MCF-7 Pre Post Pre Post Pre Post 1:200

Group # Antigen IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM

1. Heptavalent-KLH + Tn 0 0 57440  640 0 0 25600  640 9.89% 9.78% 95.87% 77.77% 78.03% 16.09% AVERAGE 10 ug QS21 Tf 0 0  10240++ 260 0 0 10240+ 160 9.39% 9.94% 96.40% 80.96% 79.70% 8.16% MEDIAN sTn 0 0 3520 580 0 0 1280 320 Muc1-G5 0 0 1280 80 0 0 1280 80 +CONTROL Ley 0 32 1920 2560 0 0 1280 1280 VK-9 40.08% globo H 0 48  672 128 0 80  640 160 MLS128 97.56% GM2 antiGM2 #### MBr-1 #### 2. Heptavalent-KLH + Tn 0 0 19200  840 0 0 25600  320 10.15% 10.36% 92.97% 79.87% 66.63% 28.35% 100 ug GPI100 Tf 0 0 10240+ 260 0 0 10240+ 160 10.10% 10.35% 95.99% 93.53% 69.88% 26.96% sTn 0 0  720 360 0 0  320 80 Muc1-G5 0 0 1520 80 0 0 2560 160 Ley 0 0 3920 680 0 0 5120 640 globo H 0 0  320 176 0 0  80 160 GM2 3. Heptavalent-KLH + Tn 0 0 32000  480 0 0 25600  320 10.29% 10.14% 92.85% 62.94% 65.36% 18.34% 100 ug GPI- Tf 0 0 10240+ 340 0 0 10240+ 320 10.20% 10.23% 92.35% 58.56% 63.59% 14.46% 100 (Lyophilized) sTn 0 0 1200 260 0 0 1280 320 Muc1-G5 0 0 1120 0 0 0  320 0 Ley 0 0  320 720 0 0  480 640 globo H 0 0  128 440 0 0   0 200 GM2 4. Heptavalent-KLH + Tn 0 0 38400  400 0 0 25600  160 10.14% 10.18% 91.34% 57.09% 70.79% 6.70% 100 ug GPI100′ + Tf 0 0 10240+ 140 0 0 10240+ 160 10.22% 10.29% 90.80% 61.70% 67.27% 4.98% polysorbate80 sTn 0 0 1960 200 0 0 1280 320 Muc1-G5 0 0 4000 20 0 0 2560 0 Ley 0 0 1440 960 0 0 1280 640 globo H 0 40  100 224 0 40  120 160 GM2 5. Heptavalent-KLH + Tn 0 0 61400  808 0 0 25600  640 9.92% 10.30% 89.55% 72.17% 64.38% 12.43% 100 ug GPI100 Tf 0 0  10240++ 560 0 0  10240++ 640 10.19% 10.19% 90.87% 70.90% 66.42% 14.32% Cytoxan 25 mg/Kg (I.P.) Day - 1 sTn 0 0  520 480 0 0  640 320 Muc1-G5 0 0 3840 0 0 0 2560 0 Ley 0 0  480 200 0 0   0 80 globo H 0 0  32 540 0 0   0 400 6. Heptavalent-KLH + Tn 0 0 32000  250 0 0 25600  160 10.33% 10.08% 90.59% 65.89% 62.40% 17.62% 100 ug GPI100 Tf 0 0 10240+ 1040 0 0 10240+ 1280 10.26% 10.01% 91.50% 62.97% 63.04% 8.00% mAb CTLA-4 in vaccine sTn 0 0 6240 180 0 0 1280 200 (100 ug/mice) Day 0 Muc1-G5 0 0 1950 40 0 0 2560 40 Day 7 & 14 no CTLA-4 Ley 0 0  180 2600 0 0  40 40 globo H 0 0   0 240 0 0   0 240 GM2 7. Heptavalent-KLH + Tn 0 0 32000  1600 0 0 25600  1280 10.05% 10.31% 90.31% 85.42% 67.29% 29.21% 100 ug GPI100 Tf 0 0 10240+ 1920 0 0 10240+ 1280 9.64% 10.41% 90.01% 85.58% 69.08% 21.26% mAb CTLA-4 in vaccine sTn 0 0 2880 100 0 0 2560 80 (100 ug/mice) Day 0 & 7 Muc1-G5 0 0 6400 60 0 0 5120 80 Day 14 no CTLA-4 Ley 0 0  32 208 0 0   0 160 globo H 0 0  32 220 0 0   0 240 GM2 8. Heptavalent-KLH + Tn 0 0 85333  40 0 0 51200+ 40 9.69% 10.15% 91.29% 45.33% 63.00% 6.19% 100 ug GPI100 Tf 0 0  10240++ 370 0 0  10240++ 320 9.63% 10.09% 90.20% 45.21% 50.05% 7.00% mAb CTLA-4 not in vaccine sTn 0 0  480 0 0 0  640 0 I.P. day - 1, 0, 1 Muc1-G5 0 0 3413 0 0 0 2560 0 Ley 0 0  320 106 0 0  160 0 globo H 0 0  53 240 0 0   0 80 GM2 9. Heptavalent-KLH + Tn 0 0 56320  640 0 0 51200  640 10.25% 10.13% 93.16% 90.64% 73.15% 45.88% 100 ug GPI100 Tf 0 0  10240+++ 2720 0 0  10240+++ 2560 10.27% 10.21% 94.17% 95.54% 76.62% 30.38% Control mAb ROR-g2 sTn 0 0 3040 5200 0 0 1280 320 100 ug/mice Muc1-G5 0 0 10240  160 0 0 10240  160 I.P. day - 1.0.1 Ley 0 0  144 464 0 0  80 160 globo H 0 0  112 848 0 0  80 640 GM2 10. Heptavalent-KLH + Tn 0 0 81920  320 0 0 51200  160 10.42% 9.98% 88.03% 76.96% 67.02% 33.26% 100 ug GPI100 Tf 0 0  10240++ 920 0 0  10240++ 320 10.50% 10.01% 87.97% 90.19% 64.58% 24.15% 100 ug/mice sTn 0 0 2240 1440 0 0 2560 160 Muc1-G5 0 0 4160 40 0 0 2560 0 Ley 0 144  144 2640 0 160  80 320 globo H 0 0   0 224 0 0   0 160 GM2

Example 2

A Preclinical Study Comparing Approaches for Augmenting the Immunogenicity of a Heptavalent KLH-Conjugate Vaccine Against Epithelial Cancers

Previously using a series of monovalent vaccines, we have demonstrated that the optimal method for inducing an antibody response against cancer cell-surface antigens is covalent conjugation of the antigens to keyhole limpet hemocyanin (KLH) and the use of a saponin adjuvant. In preparation for testing a polyvalent (heptavalent)-KLH conjugate vaccine in the clinic, we have tested the impact on antibody induction against the 7 antigens of several variables described by others to augment immunogenicity. We explore here the impact of approaches for decreasing suppression of the immune response (low dose cyclophosphamide and anti-CTLA4 mAb), different saponin adjuvants (QS-21 and GPI-0100), and different methods of formulation (lyophilization and use of polysorbate 80). After two sets of experiments, these results are clear:

1) Immunization with the heptavalent-KLH conjugate vaccine induces high titers of antibodies against Tn (median ELISA titer IgM/IgG 320/10,240), sTn (640/2560), TF (320/5120), MUC1 (80/20,480) and globo H (1280/10), lower titers of antibodies against Lewis Y (160/80) and only occasional antibodies against GM2. 2) These antibodies reacted with the purified synthetic antigens by ELISA, and with naturally expressed antigens on the cancer cell surface by FACS. 3) Neither decreasing suppression with low dose cyclophosphamide or anti-CTLA4 mAb, nor changing the standard formulation by lyophilization or use of polysorbate 80 had any impact on antibody titers. 4) The two saponin adjuvants were comparably potent at our standard doses (QS-21 10 ug and GPI-0100 100 ug) but a third experiment comparing higher doses is in progress.

The high titers of antibodies against this heptavalent vaccine and the inability of these additional approaches to further augment antibody titers confirms that the combination of conjugation to KLH and use of a saponin adjuvant is sufficiently optimized for testing in the clinic.

There is a broad and expanding body of pre-clinical and clinical studies demonstrating that naturally acquired, actively induced, and passively administered antibodies are able to eliminate circulating tumor cells and micro metastases (1). Induction of antibodies against tumor antigens is more difficult than induction of antibodies against viral and bacterial antigens because most tumor antigens are normal or slightly modified auto antigens and because actively growing tumors may set in motion mechanisms which suppress the anti-cancer cell immune response. Consequently it may be necessary to overcome not only some level of tolerance but also some additional level of active suppression, making the immunization approach critical. We have previously reported that the optimal approach for induction of antibodies against gangliosides and a variety of other carbohydrate and peptide antigens is covalent attachment of the tumor antigen to an immunogenic carrier molecule (keyhole limpet hemocyanin (KLH) was optimal (2,3)) plus the use of a potent immunological adjuvant. In our previous experience saponin adjuvants such as QS-21 and GPI-0100 were the optimal adjuvants (4,5).

In preparation for clinical trials with a heptavalent KLH-conjugate vaccine we test here the impact of several variables including 1) vaccine formulation (lyophilization or the use of polysorbate 80), 2) decreasing suppression (low dose cyclophosphamide or anti-CTLA4 mAb), or 3) various doses of the two saponin adjuvants QS-21 and GPI-0100, on antibody titers against the individual antigens and tumor cells expressing these antigens.

Pathogen-free female BALB/c or C57BL/6 mice 6-10 weeks of age were obtained from the Jackson Laboratory (Bar Harbor, Me.). QS-21 was obtained from Aquila Biopharmaceuticals (Framingham, M A (now Antigenics Inc., NYC, NY)), GPI-0100 was obtained from Galenica Pharmaceuticals, Inc. (Birmingham, Ala.). Cytoxan (25 mg/kg) was purchased and injected IP one day prior to the first immunization. The hybridoma for murine monoclonal antibody CTLA-4 was obtained from Jim Allison (Berkeley, Calif.) and the mAb was prepared by Dr. Polly Gregor (MSKCC). The reactivity of mAb with CTLA-4 was confirmed. Polysorbate 80 was purchased.

Immunization of mice: groups of five mice were immunized 3 times at one week intervals with the heptavalent vaccine containing 3 mcg of each of the 7 antigens covalently conjugated to KLH and mixed with GPI-0100 or QS-21 as indicated. Vaccines were administered subcutaneously over the lower abdomen. A 4th, booster, immunization was given at week 8.

Serological Assays For the ELISA assay, glycosylated MUC1, globo H, Lewis Y or GM2, or Tn, sTn or TP conjugated to BSA, were coated on ELISA plates at an antigen dose of 0.1-0.2 mcg per well.

Phosphatase-conjugated goat anti-mouse IgG or IgM was added at a dilution of 1:200 (Southern Biotechnology Associates, Inc., Birmingham, Ala.). Antibody titer was the highest dilution yielding absorbance of 0.10 or greater.

F ACS analysis: MCF-7 human breast cancer cells expressing all seven antigens but especially Lewis Y and MUC1 and sTn, and LSC expressing especially Lewis Y, sTn and Tn, were used. Single cell suspensions of 5×10⁷ cells/tube were washed in PBS with 3% fetal calf serum and incubated with 20 mcl of full strength or 1/200 diluted antisera for 30 minutes on ice. 20 microliters of 1/15 goat anti-mouse IgG or IgM labeled with FITC were all added and percent positive cells and mean fluorescent intensity (MFI) of stained cells analyzed using a F ACScan (Becton Dickenson, Calif.). Pre and post vaccination sera were analyzed together and the pre-treatment percent positive cells set at 10%. Comparison of the Immune Response after Immunization with Monovalent and Hexavalent-KLH Conjugate Vaccines Against Prostate Cancer

Glycolipid and glycoprotein differentiation antigens such as GM2, Globo H, Lewis y, Tn, TF, and mucin 1 (MUC1) are over-expressed on the cell surface of many tumors. Of the many approaches to immunization we have tested, covalent conjugation of antigens such as these to keyhole limpet hemocyanin (KLH) plus the use of immunological adjuvant QS-21 has been the optimal approach for inducing IgM and IgG antibodies. Immunization of patients with monovalent vaccines containing these antigens has demonstrated the consistent immunogenicity and safety of these vaccines. However, to overcome the heterogeneous nature of tumors, and of the immune response in different individuals, we have recently vaccinated a small group of patients (prostate cancer with rising PSA, but free of detectable disease) with a hexavalent-KLH vaccine containing GM2, Globo H, Le^(y), Tn(c), TF(c) and glycosylated MUC1 individually conjugated with KLH and mixed with immunological adjuvant QS-21. The main objective of this presentation is to compare the immune response of the six initial patients receiving hexavalent vaccine with the immune responses of patients who had previously been immunized with the respective monovalent vaccines. All patients were vaccinated six times (weeks 1, 2, 3, 7, 19 and 31) and bloods obtained pre treatment and on weeks 7 and 9 were tested at one time. RECIPRICOL MEAN PEAK ELISA TITER AFTER IMMUNIZATION IgM/IgG.

Antigen GM2 Globo H Le^(y) Tn TF MUC1 Poly- Pending 160/0 0/0  640/640 1280/160  40/320 valent vaccine Indivi- Pending 160/0 0/10 1280/2560 1280/160 2560/320  dual Vaccine

Because of the low response against Le^(y), we are continuing studies aimed at creating a more immunogenic Le^(y) vaccine. Comparing the responses induced by monovalent and hexavalent vaccines, there was no significant difference in the antibody responses against any of the five antigens tested to date. Combination of six individual conjugates into a single vaccine does not significantly change the antibody response against the individual antigens.

Experiment 1: Median ELISA titers and FACS results after vaccination of groups of 5 Balb/c mice with Heptavalent-KLH conjugate ELISA(mean) ELISA(median) Pre Post Pre Post Group # Antigen IgG IgM IgG IgM IgG IgM IgG IgM 1. Heptavalent-KLH + Tn 0 0 57440  640 0 0 25600  640 10 ug QS21 Tf 0 0  10240++ 260 0 0 10240+ 160 sTn 0 0 3520 580 0 0 1280 320 Muc1- 0 0 1280 80 0 0 1280 80 G5 Ley 0 32 1920 2560 0 0 1280 1280 globo H 0 48  672 128 0 80  640 160 GM2 2. Heptavalent-KLH + Tn 0 0 19200  840 0 0 25600  320 100 ug GPI100 Tf 0 0 10240+ 260 0 0 10240+ 160 sTn 0 0  720 360 0 0  320 80 Muc1- 0 0 1520 80 0 0 2560 160 G5 Ley 0 0 3920 680 0 0 5120 640 globo H 0 0  320 176 0 0  80 160 GM2 3. Heptavalent-KLH + Tn 0 0 32000  480 0 0 25600  320 100 ug GPI-100 Tf 0 0 10240+ 340 0 0 10240+ 320 (Lyophilized) sTn 0 0 1200 260 0 0 1280 320 Muc1- 0 0 1120 0 0 0  320 0 G5 Ley 0 0  320 720 0 0  480 640 globo H 0 0  128 440 0 0   0 200 GM2 4. Heptavalent-KLH + Tn 0 0 38400  400 0 0 25600  160 100 ug GPI100′ + Tf 0 0 10240+ 140 0 0 10240+ 160 polysorbate80 sTn 0 0 1960 200 0 0 1280 320 Muc1- 0 0 4000 20 0 0 2560 0 G5 Ley 0 0 1440 960 0 0 1280 640 globo H 0 40  100 224 0 40  120 160 GM2 5. Heptavalent-KLH + Tn 0 0 61400  808 0 0 25600  640 100 ug GPI100 Tf 0 0  10240++ 560 0 0  10240++ 640 Cytoxan 25 mg/Kg sTn 0 0  520 480 0 0  640 320 (I.P.) Day −1 Muc1- 0 0 3840 0 0 0 2560 0 G5 Ley 0 0  480 200 0 0   0 80 globo H 0 0  32 540 0 0   0 400 GM2 6. Heptavalent-KLH + Tn 0 0 32000  250 0 0 25600  160 100 ug GPI100 Tf 0 0 10240+ 1040 0 0 10240+ 1280 mAb CTLA-4 in sTn 0 0 6240 180 0 0 1280 200 vaccine Muc1- 0 0 1950 40 0 0 2560 40 (100 ug/mice) Day 0 G5 Day 7 & 14 no Ley 0 0  180 2600 0 0  40 40 CTLA-4 globo H 0 0   0 240 0 0   0 240 GM2 7. Heptavalent-KLH + Tn 0 0 32000  1600 0 0 25600  1280 100 ug GPI100 Tf 0 0 10240+ 1920 0 0 10240+ 1280 mAb CTLA-4 in sTn 0 0 2880 100 0 0 2560 80 vaccine Muc1- 0 0 6400 60 0 0 5120 80 (100 ug/mice) Day 0 G5 & 7 Ley 0 0  32 208 0 0   0 160 Day 14 no CTLA-4 globo H 0 0  32 220 0 0   0 240 GM2 8. Heptavalent-KLH + Tn 0 0 85333  40 0 0 51200+ 40 100 ug GPI100 Tf 0 0  10240++ 370 0 0  10240++ 320 mAb CTLA-4 not in sTn 0 0  480 0 0 0  640 0 vaccine Muc1- 0 0 3413 0 0 0 2560 0 I.P. day −1, 0, 1 G5 Ley 0 0  320 106 0 0  160 0 globo H 0 0  53 240 0 0   0 80 GM2 9. Heptavalent-KLH + Tn 0 0 56320  640 0 0 51200  640 100 ug GPI100 Tf 0 0  10240+++ 2720 0 0  10240+++ 2560 Control mAb ROR- sTn 0 0 3040 5200 0 0 1280 320 g2 Muc1- 0 0 10240  160 0 0 10240  160 100 ug/mice G5 I.P. day −1, 0, 1 Ley 0 0  144 464 0 0  80 160 globo H 0 0  112 848 0 0  80 640 GM2 10. Heptavalent- Tn 0 0 81920  320 0 0 51200  160 KLH + Tf 0 0  10240++ 920 0 0  10240++ 320 100 ug GPI100 sTn 0 0 2240 1440 0 0 2560 160 100 ug/mice Muc1- 0 0 4160 40 0 0 2560 0 G5 Ley 0 144  144 2640 0 160  80 320 globo H 0 0   0 224 0 0   0 160 GM2 FACS MCF-7 Pre Post 1:200 Group # Antigen IgG IgM IgG IgM IgG IgM +CONTROL 1. Heptavalent-KLH + Tn 10% 10% 96% 78% 78% 16% VK-9 40.08% 10 ug QS21 Tf  9% 10% 96% 81% 80%  8% MLS128 97.56% sTn antiGM2 98.87% Muc1- MBr-1 58.06% G5 Ley globo H GM2 2. Heptavalent-KLH + Tn 10% 10% 93% 80% 67% 28% 100 ug GPI100 Tf 10% 10% 96% 94% 70% 27% sTn Muc1- G5 Ley globo H GM2 3. Heptavalent-KLH + Tn 10% 10% 93% 63% 65% 18% 100 ug GPI-100 Tf 10% 10% 92% 59% 64% 14% (Lyophilized) sTn Muc1- G5 Ley globo H GM2 4. Heptavalent-KLH + Tn 10% 10% 91% 57% 71%  7% 100 ug GPI100′ + Tf 10% 10% 91% 62% 67%  5% polysorbate80 sTn Muc1- G5 Ley globo H GM2 5. Heptavalent-KLH + Tn 10% 10% 90% 72% 64% 12% 100 ug GPI100 Tf 10% 10% 91% 71% 66% 14% Cytoxan 25 mg/Kg sTn (I.P.) Day −1 Muc1- G5 Ley globo H GM2 6. Heptavalent-KLH + Tn 10% 10% 91% 66% 62% 18% 100 ug GPI100 Tf 10% 10% 92% 63% 63%  8% mAb CTLA-4 in sTn vaccine Muc1- (100 ug/mice) Day 0 G5 Day 7 & 14 no Ley CTLA-4 globo H GM2 7. Heptavalent-KLH + Tn 10% 10% 90% 85% 67% 29% 100 ug GPI100 Tf 10% 10% 90% 86% 69% 21% mAb CTLA-4 in sTn vaccine Muc1- (100 ug/mice) Day 0 G5 & 7 Ley Day 14 no CTLA-4 globo H GM2 8. Heptavalent-KLH + Tn 10% 10% 91% 45% 63%  6% 100 ug GPI100 Tf 10% 10% 90% 45% 50%  7% mAb CTLA-4 not in sTn vaccine Muc1- I.P. day −1, 0, 1 G5 Ley globo H GM2 9. Heptavalent-KLH + Tn 10% 10% 93% 91% 73% 46% 100 ug GPI100 Tf 10% 10% 94% 96% 77% 30% Control mAb ROR- sTn g2 Muc1- 100 ug/mice G5 I.P. day −1, 0, 1 Ley globo H GM2 10. Heptavalent- Tn 10% 10% 88% 77% 67% 33% KLH + Tf 11% 10% 88% 90% 65% 24% 100 ug GPI100 sTn 100 ug/mice Muc1- G5 Ley globo H GM2

Experiment 2: Median ELISA titers and FACS results against MCF-7 and LSC cells after vaccination groups of 5 C57BL/6 Mice with heptavalent-KLH conjugate vaccine FACS with LSC ELISA FACS withMCF-7 # LSC Pre Post Pre Post 1:200 Pre Post 1:200 Group # Antigen IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM Heptavalent- Tn 0 10 5120+ 320 10% 11% 91.61% 18.46% 80% 18% 10% 11% 96.04% 39.25% 84% 39% KLH + Tf 0 10 5120 320   99%   24% 82% 21% 100 ug sTn 0 0 2560 640 GPI100 Muc1- 0 0 5120++ 80 (200 ul/ G5 mice) Ley 0 0  80 160 globo H 0 80  10 1280 GM2 0 0   0 10 Heptavalent- Tn 0 0 2560 40 11% 11% 84.31%  9.89% 60% 10% 10% 10% 92.22% 16.68% 63% 17% KLH + Tf 0 0 5120 40 100 ug GPI sTn 0 0  320 160 (old batch) Muc1- 0 0 1280 0 (200 ul/ G5 mice) Ley 0 0  40 320 globo H 0 80   0 320 GM2 0 0   0 0 Heptavalent- Tn 0 0 5120+ 640 11% 10% 90.55% 12.59% 75% 13% 11% 10% 85.22% 11.17% 54% 11% KLH + Tf 0 0 5120 320 100 ug sTn 0 0  640 640 GPI-100 + Muc1- 0 0 5120 80 polysorbate G5 80 (200 ul/ Ley 0 0   0 320 mice) globo H 0 160  20 1280 GM2 0 0   0 0 Heptavalent- Tn 0 20 5120+ 160 11% 10% 90.96%  4.8% 71% 5% 10% 11% 70.28%  8.33% 41%  8% KLH + Tf 0 80 5120+ 640 10 ug QS-21 sTn 0 0 2560 320 (200 ul/ Muc1- 0 0 2560 160 mice) G5 Ley 0 0  10 10 globo H 0 80  40 640 GM2 0 0   0 0 Heptavalent- Tn 0 20  160 160 11% 11% 60.86% 12.60% 16% 13% 11% 10% 45.30% 11.86%  6% 12% KLH + Tf 0 10  320 160 10 ug sTn 0 40  20 160 ER803732 Muc1- 0 0  160 80 (200 ul/ G5 mice) Ley 0 0   0 80 globo H 20 80  80 320 GM2 0 0   0 0

Protocol 00-106: Pilot Phas Trial: Vaccination of Patients Who Have Ovarian, Fallopian Tube or Peritoneal Cancer with A Polyvalent Vaccine-KLH Conjugate + QS-21 Vaccine:  10ug GM2  10ug Globo-H  10ug LeY  3ug Muc1G5  3ug Tn(c)  3ug S-Tn(c)  3ug TF(c) Patient Name Muc1-IG5 Globo-H LeY GM2 Tn(c) S-Tn(c) Tf(c) Patient # Vaccination Serology Sera # IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG Jun. 21, Jun. 21, Jun. 24, Jun. 24, Jun. 25, Jun. 25, Jun. 12, 2002 Jun. 12, 2002 Jun. 27, 2002 Jun. 19, 2002 2002 2002 2002 2002 2002 2002 Patient 1 Jul. 03, 2001 Jul. 03, 2001 P7OVQ 2 0 0 0 0 0 0 0 0 0 20 Jul. 10, 2001 Jul. 31, 2001 P7OVQ 6 2560 640 40 0 0 0 0 20 1280 160 Jul. 17, 2001 Aug. 14, 2001 P7OVQ 9 2560 640 40 0 0 0 0 20 1280 160 Aug. 14, 2001 Aug. 08, 2001 P7OVQ 15 2560 320 40 0 0 0 0 20 1280 320 OFF TRIAL (+) Control M62 2560 640 Positive control 2560 S193 (1 mg/ml) 6400 Positive control 2560 Positive control 640 Positive control 2560 Positive control 2560 2560 Positive control 2560 Jun. 04, 2002 Jun. 04, 2002 Jun. 27, 2002 Jun. 26, 2002 Patient 2 Jul. 03, 2001 Jul. 03, 2001 P7OVQ 3 0 0 0 0 Jul. 10, 2001 Jul. 31, 2001 P7OVQ 7 80 20 0 0 Jul. 17, 2001 Aug. 14, 2001 P7OVQ 10 40 20 0 0 Aug. 14, 2001 Aug. 28, 2001 P7OVQ 13 20 40 0 0 Sep. 25, 2001 P7OVQ 20 20 20 0 0 Oct. 09, 2001 Oct. 09, 2001 P7OVQ 24 20 20 0 0 Oct. 23, 2001 P7OVQ 30 20 20 0 0 Jan. 11, 2002 P7OVQ 49 0 0 0 0 Jun. 07, 2002 P7OVQ 65 0 0 (+) Control Positive control 2560 640 Positive control 2560 Positive control 640 Patient Name Muc1-IG5 Globo-H LeY GM2 Tn(c) STn(c) Tf Medical # Vaccination Serology Sera # IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG Jun. 07, 2002 Jun. 07, 2002 Jun. 27, 2002 Jun. 26, 2002 Patient 3 Jul. 10, 2001 Jul. 10, 2001 P7OVQ 4 0 0 20 0 Jul. 17, 2001 Aug. 07, 2001 P7OVQ 8 1280 160 160 0 Jul. 24, 2001 Aug. 21, 2001 P7OVQ 12 320 40 80 0 Aug. 21, 2001 Sep. 04, 2001 P7OVQ 18 160 80 80 0 Oct. 02, 2001 P7OVQ 22 80 40 40 0 Oct. 16, 2001 Oct. 16, 2001 P7OVQ 26 40 40 80 0 Oct. 30, 2001 P7OVQ 33 80 80 40 0 Oct. 08, 2002 P7OVQ 47 40 40 80 0 (+) Control M62 2560 1280 Positive control 2560 Positive control 1280 Aug. 10, 2002 Jun. 10, 2002 patient 4 Jul. 24, 2001 Jul. 24, 2001 P7OVQ 5 0 0 Jul. 31, 2001 Aug. 21, 2001 P7OVQ 14 1280 80 Aug. 07, 2001 Sep. 04, 2001 P7OVQ 17 1280 40 Sep. 04, 2001 Sep. 18, 2001 P7OVQ 23 160 20 Oct. 16, 2001 P7OVQ 27 160 20 Oct. 30, 2001 Oct. 30, 2001 P7OVQ 32 60 40 Nov. 13, 2001 P7OVQ 36 20 20 Jan. 22, 2002 P7OVQ 52 20 20 Mar. 26, 2002 P7OVQ 63 20 20 (+) Control M62 1280 640 Jun. 21, Jun. 21, Jun. 24, Jun. 24, Jun. 25, Jun. 25, Jun. 12, 2002 Jun. 12, 2002 Jun. 27, 2002 Jun. 19, 2002 2002 2002 2002 2002 2002 2002 patient 5 Aug. 16, 2001 Aug. 16, 2001 P7OVQ 11 0 0 0 0 0 0 0 0 0 0 Aug. 23, 2001 Sep. 27, 2001 P7OVQ 21 80 80 0 0 0 0 0 0 40 160 Aug. 30, 2001 Oct. 11, 2001 P7OVQ 25 40 80 0 0 0 0 0 0 0 160 Sep. 27, 2001 OFF TRIAL (+) Control M62 2560 640 Positive control 2560 S193 (1 mg/ml) 6400 Positive control 2560 Positive control 640 Positive control 2560 Positive control 2560 2560 Positive control 2560 Jun. 17, 2002 Jun. 17, 2002 patient 6 Aug. 30, 2001 Aug. 30, 2001 P7OVQ 18 0 0 Sep. 06, 2001 Sep. 27, 2001 P7OVQ 19 160 20 Sep. 13, 2001 Oct. 25, 2001 P7OVQ 31 40 20 Oct. 11, 2001 Nov. 22, 2001 P7OVQ 39 20 0 Dec. 06, 2001 Dec. 06, 2001 P7OVQ 41 20 0 Dec. 20, 2001 P7OVQ 44 20 20 Mar. 08, 2002 P7OVQ 62 20 20 (+) Control M62 2560 320 Jun. 21, Jun. 21, Jun. 24, Jun. 24, Jun. 25, Jun. 25, Jun. 13, 2002 Jun. 13, 2002 Jun. 27, 2002 Jun. 19, 2002 2002 2002 2002 2002 2002 2002 patient 7 Oct. 19, 2001 Oct. 19, 2001 P7OVQ 28 0 0 0 (re-do) 0 0 0 0 20 0 Oct. 26, 2001 Nov. 16, 2001 P7OVQ 35 640 40 0 (re-do) 0 0 0 0 160 160 Nov. 02, 2001 Dec. 14, 2001 P7OVQ 42 80 20 0 (re-do) 0 0 0 0 80 160 Nov. 30, 2001 Jan. 11, 2002 P7OVQ 48 80 20 0 (re-do) 0 0 0 20 80 80 Jan. 25, 2002 Jan. 25, 2002 P7OVQ 53 80 20 0 (re-do) 0 0 0 20 80 40 Feb. 08, 2002 P7OVQ 56 80 20 0 (re-do) 0 0 0 20 80 80 Apr. 19, 2002 (+) Control M62 2560 160 Positive control 2560 S193 (1 mg/ml) 6400 Positive control 2560 Positive control 640 Positive control 2560 Positive control 2560 2560 Positive control 2560 Jun. 18, 2002 Jun. 18, 2002 Patient 8 Oct. 23, 2001 Oct. 23, 2001 P7OVQ 29 0 0 Oct. 30, 2001 Nov. 20, 2001 P7OVQ 38 0 0 Nov. 06, 2001 Dec. 18, 2001 P7OVQ 43 0 0 Dec. 04, 2001 Jan. 15, 2002 P7OVQ 51 0 0 Jan. 29, 2002 Jan. 29, 2002 P7OVQ 54 40 0 OFF TRIAL (+) Control M62 2560 640 Jun. 18, 2002 Jun. 18, 2002 Patient 9 Nov. 06, 2001 Nov. 08, 2001 P7OVQ 34 0 0 Nov. 13, 2001 Dec. 04, 2001 P7OVQ 40 1280 80 Nov. 20, 2001 Jan. 01, 2002 P7OVQ 45 320 80 Dec. 18, 2001 Jan. 29, 2002 P7OVQ 55 320 80 Feb. 12, 2002 Feb. 12, 2002 P7OVQ 58 160 40 Feb. 26, 2002 P7OVQ 60 160 80 May 09, 2002 P7OVQ 64 40 80 (+) Control M62 2560 320 Jun. 21, Jun. 21, Jun. 24, Jun. 24, Jun. 25, Jun. 25, Jun. 13, 2002 Jun. 13, 2002 Jun. 27, 2002 Jun. 19, 2002 2002 2002 2002 2002 2002 2002 Patient 10 Nov. 20, 2001 Nov. 20, 2001 P7OVQ 37 0 0 0 (re-do) 0 0 0 0 0 0 Nov. 27, 2001 Dec. 18, 2001 P7OVQ 46 320 60 320 (re-do) 0 20 0 20 160 80 Dec. 04, 2001 Jan. 15, 2002 P7OVQ 50 320 320 320 (re-do) 0 20 0 20 80 640 Jan. 01, 2002 Feb. 12, 2002 pt no show — — — — — — — — — — Feb. 26, 2002 Feb. 26, 2002 P7OVQ 59 40 160 80 (re-do) 0 20 0 20 40 320 Mar. 12, 2002 pt no show — — — — — — — — — — May 21, 2002 pt no show — — — — — — — — — — (+) Control M62 2560 160 Positive control 2560 S193 (1 mg/ml) 6400 Positive control 2560 Positive control 640 Positive control 2560 Positive control 2560 2560 Positive control 1280 Jun. 21, Jun. 21, Jun. 24, Jun. 24, Jun. 25, Jun. 25, Jun. 12, 2002 Jun. 12, 2002 Jun. 27, 2002 Jun. 19, 2002 2002 2002 2002 2002 2002 2002 Patient 11 Mar. 07, 2001 Mar. 07, 2001 P7OVQ 1 0 0 0 0 0 0 0 20 20 0 Feb. 05, 2002 Feb. 05, 2001 P7OVQ 57 0 0 0 0 0 0 0 0 20 0 Feb. 12, 2002 Mar. 05, 2002 P7OVQ 61 0 20 0 0 0 0 0 0 40 60 Feb. 19, 2002 OFF TRIAL Mar. 19, 2002 (+) Control M62 2560 640 Positive control 2560 S193 (1 mg/ml) 6400 Positive control 2560 Positive control 640 Positive control 2560 Positive control 2560 2560 Positive control 2560 * Patient received one vaccine before protocol hold, restarted-1 year later

Protocol # 01-019: Serological analysis of Breast cancer patient vaccinated with hexavalent vaccine ELISA LeY ELISA Globo H ELISA ELISA MUC-1-1 Ceramide Ceramide ELISA Tf GM2 (10 mcg) 5G (3 mcg) (10 mcg) (10 mcg) (3 mcg) ELISA dOSM for Tn ELISA sTn Vaccine Sample (March 2002) (March 2002) (April 2002) (May 2002) (May 2002) (3 mcg) (August 2002) (3 mcg) Patient # Date Date Serology Top IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM Patient 1 Jan. 16, 2002 Jan. 16, 2002 P7BRQ4 20 0 0 0 0 0 0 0 0 20 Jan. 23, 2002 Jan. 23, 2002 P7BRQ9 0 0 0 0 0 0 0 0 0 40 Jan. 30, 2002 Jan. 30, 2002 P7BRQ11 0 0 0 0 0 0 0 20 0 40 Feb. 13, 2002 P7BRQ19 0 0 40 80 0 0 0 20 320 640 Feb. 27, 2002 Feb. 27, 2002 P7BRQ28 0 0 20 80 0 0 0 0 80 1280 Mar. 13, 2002 P7BRQ37 0 40 40 80 0 0 0 20 160 1280 Apr. 10, 2002 P7BRQ44 80 320 0 0 0 20 80 1280 May 22, 2002 May 22, 2002 P7BRQ62 40 640 Jun. 05, 2002 P7BRQ67 Controls >2560 (M62) 320 320 2560 (MCG170) 160 2560 (LeYM12) 20 80/160 Mono Ab (S193) 1280 (GB81) 0 320 Mono Ab (VK9) 5120 (P7BRQ17) 320 >2560 (slovin lab wk7) Patient 2 Jan. 16, 2002 Jan. 16, 2002 P7BRQ5 0 0 0 0 0 0 0 20 20 Jan. 23, 2002 Jan. 23, 2002 P7BRQ8 0 0 0 0 0 0 0 40 0 40 Jan. 30, 2002 Jan. 30, 2002 P7BRQ12 0 0 0 0 0 0 0 2560 160 640 Feb. 13, 2002 P7BRQ20 0 0 80 160 0 0 0 640 160 1280 Feb. 27, 2002 Feb. 27, 2002 P7BRQ27 0 80 40 20 0 0 0 640 160 1280 Mar. 13, 2002 P7BRQ36 0 160 80 20 0 0 0 320 320 640 Apr. 10, 2002 P7BRQ46 80 80 0 0 0 80 160 640 May 22, 2002 May 22, 2002 P7BRQ61 160 640 Jun. 05, 2002 P7BRQ68 Controls >2560 (P7BRQ17) 320 >2560 (hexavalent) 80 320 (P7BRQ16) 640 (LeYM12) 0 150 Mono Ab (S193) 1280 (GB81) 0 320 Mono Ab (VK9) 5120 (P7BRQ17) 320 >2560 (slovin lab wk7) Patient 3 Jan. 07, 2002 Jan. 07, 2002 P7BRQ1 0 0 0 0 0 0 0 0 0 0 Jan. 14, 2002 Jan. 14, 2002 P7BRQ3 0 0 0 0 0 0 0 0 0 0 Jan. 21, 2002 Jan. 21, 2002 P7BRQ7 0 0 0 320 0 20 0 320 0 80 Feb. 04, 2002 P7BRQ17 0 20 80 2560 0 40 0 640 320 2560 Feb. 18, 2002 Feb. 18, 2002 P7BRQ23 0 0 80 640 0 20 0 320 320 1280 Mar. 04, 2002 P7BRQ30 0 0 80 320 0 0 0 320 1280 640 Apr. 01, 2002 P7BRQ41 80 80 0 0 0 80 320 320 May 13, 2002 May 13, 2002 P7BRQ57 0 20 160 320 May 27, 2002 P7BRQ63 0 20 Controls >2560 (P7BRQ17) 320 (hexavalent) 160 1280 (P7BRQ16) 320 2560 (LeYM12) 20 160 Mono Ab (S193) 1280 (GB81) 0 320/640 Mono Ab (VK9) 0 1280/2560 (P7BRQ17) 320 2560 (slovin lab wk7) ELISA ELISA MUC-1-1 ELISA GM2 5G ELISA LeY Globo H ELISA Tf ELISA (10 mcg) (3 mcg) Ceramide Ceramide (10 mcg) dOSM for ELISA (March (March (10 mcg) (10 mcg) (May Tn (3 mcg) sTn Patient Vaccine Sample 2002) 2002) (April 2002) (May 2002) 2002) (June 2002) (3 mcg) Name Date Date Serology Top IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM Patient 4 Jan. 14, 2002 Jan. 14, 2002 P7BRQ2 0 0 0 0 0 40 0 20 0 40 Jan. 21, 2002 Jan. 21, 2002 P7BRQ6 0 0 0 0 0 20 20 80 0 20 did not Jan. 28, 2002 Jan. 28, 2002 P7BRQ10 0 0 0 80 0 40 0 320 20 160 come in Feb. 11, 2002 P7BRQ16 0 0 640 320 0 1280 0 640/1280 40 320 Feb. 25, 2002 Feb. 25, 2002 P7BRQ26 0 0 160 40 0 320 0 320 40 640 Mar. 11, 2002 P7BRQ34 0 0 160 20 0 640 80 320 160 160 Apr. 08, 2002 Apr. 15, 2002 P7BRQ47 320 80 0 320 0 160 160 160 May 20, 2002 May 20, 2002 P7BRQ58 0 40 320 160 Jun. 03, 2002 P7BRQ65 80 40 Controls >2560 (P7BRQ17) 320 (hexavalent) 320 >2560 (P7BRQ16) 160 >2560 (LeYM12) 20 160 Mono Ab (S193) 1280 (GB81) 20 320/640 Mono Ab (VK9) 0 1280/2560 (P7BRQ17) 320 >2560 (slovin lab wk7) Patient 5 Feb. 06, 2002 Feb. 06, 2002 P7BRQ15 0 0 0 0 0 0 0 0 0 0 Feb. 13, 2002 Feb. 13, 2002 P7BRQ22 0 0 0 20 0 0 0 0 0 0 Feb. 20, 2002 Feb. 20, 2002 P7BRQ25 0 0 0 0 0 0 0 0 0 0 Mar. 06, 2002 P7BRQ33 0 0 40 40 0 0 0 20 20 40 Mar. 20, 2002 Mar. 20, 2002 P7BRQ38 0 0 20 40 0 0 0 0 0 40 Apr. 03, 2002 P7BRQ42 160 40 0 0 0 0 40 40 May 01, 2002 P7BRQ51 0 20 20 20 Jun. 12, 2002 Jun. 12, 2002 P7BRQ71 Jun. 26, 2002 Controls >2560 (P7BRQ17) 320 80 160 (hexavalent) 640 320 (P7BRQ16) 20 160 (LeYM12) 1280 Mono Ab (S193) 0 320/640 (GB81) 0 1280/2560 Mono Ab (VK9) 320 >2560 (P7BRQ17) 320 1280 (solvin tab wk7) Patient 6 Feb. 06, 2002 Feb. 06, 2002 P7BRQ14 0 20 0 0 20 0 0 0 0 0 Feb. 13, 2002 Feb. 13, 2002 P7BRQ18 0 20 0 0 0 0 0 0 0 0 Feb. 20, 2002 Feb. 20, 2002 P7BRQ24 0 40 0 80 0 0 0 160 160 80 Mar. 06, 2002 P7BRQ31 0 20 320 320 0 0 0 160 160 320 Mar. 20, 2002 Mar. 20, 2002 P7BRQ39 0 20 160 320 0 0 0 80 40 160 Apr. 03, 2002 P7BRQ43 320 40 0 0 0 40 160 160 May 01, 2002 P7BRQ52 0 40 320 320 Jun. 12, 2002 Jun. 12, 2002 P7BRQ70 Jun. 26, 2002 Controls >2560 (P7BRQ17) 320 80 640 (hexavalent) 640 320 (P7BRQ16) 0 80/160 (LeYM12) 1280 Mono Ab (S193) 0 320/640 (GB81) 0 1280/2560 Mono Ab (VK9) 320 2560 (P7BRQ17) 320 1280 (solvin tab wk7) ELISA ELISA ELISA MUC-1-1 ELISA LeY Globo H ELISA Tf ELISA GM2 5G (3 mcg) Ceramide Ceramide (10 mcg) dOSM for ELISA (10 mcg) (April (10 mcg) (10 mcg) (May Tn (3 mcg) sTn Patient Vaccine Sample (April 2002) 2002) (April 2002) (May 2002) 2002) (June 2002) (3 mcg) Name Date Date Serology Top IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM Patient 7 Feb. 27, 2002 Feb. 27, 2002 P7BRQ29 0 0 0 0 0 0 0 0 0 20 Mar. 06, 2002 Mar. 06, 2002 P7BRQ32 0 0 0 0 0 0 0 0 0 0 Mar. 13, 2002 Mar. 13, 2002 P7BRQ35 0 20 20 320 0 0 0 0 320 160 Mar. 27, 2002 P7BRQ40 0 40 320 320 0 0 0 0 640/1280 160 Apr. 10, 2002 Apr. 10, 2002 P7BRQ45 0 80 320 640 0 0 0 0 640 80 Apr. 24, 2002 P7BRQ49 0 60 160 320 0 0 0 0 1280 80 May. 22, 2002 P7BRQ60 0 0 640 80 Jul. 03, 2002 Jul. 03, 2002 Controls >2560 (P7BRQ17) 320 80 640 (hexavalent) 640 320 0 1280 (P7BRQ16) 0 160 (LeYM12) 1280 Mono Ab (S193) 20 320 (GB81) 0 1280/2560 Mono Ab (VK9) 640 2560 (P7BRQ17) 640 1280 (solvin tab wk7) (June 2002) (June 2002) (July 2002) (July 2002) Patient 8 Apr. 22, 2002 Apr. 22, 2002 P7BRQ48 Apr. 29, 2002 Apr. 29, 2002 P7BRQ53 May 06, 2002 May 06, 2002 P7BRQ54 May 20, 2002 P7BRQ59 Aug. 03, 2002 Jun. 03, 2002 P7BRQ85 Jun. 17, 2002 P7BRQ72 Jul. 15, 2002 Aug. 26, 2002 Aug. 26, 2002 Sep. 09, 2002 Controls (P7BRQ17) (hexavalent) (P7BRQ16) (LeYM12) Mono Ab (S193) (GB81) Mono Ab (VK9) (P7BRQ17) (slovin tab wk7) Patient 9 Apr. 29, 2002 Apr. 29, 2002 P7BRQ50 May 06, 2002 May 08, 2002 P7BRQ55 May 13, 2002 May 13, 2002 P7BRQ56 May 27, 2002 P7BRQ64 Aug. 10, 2002 Jun. 10, 2002 P7BRQ69 Jun. 24, 2002 Jul. 22, 2002 Sep. 02, 2002 Sep. 02, 2002 Sep. 16, 2002 Controls (P7BRQ17) (hexavalent) (P7BRQ16) (LeYM12) Mono Ab (S193) (GB81) Mono Ab (VK9) (P7BRQ17) (slovin tab wk7) Notes Monoclonals: Dilutions VK9 1:200; S193 1:400 DOSM Tested positive by ELISA with Monoclonals 5F4 and HB-TN1. OSM Tested positive by ELISA with Monoclonals 5F4, B72.3, and HB-TN1. Shaded region denotes Pre sera (week 1) All samples were tested in duplicate. for patients Lewis and Smith, Lewis Y plates were coated at 0.3, future plates will be coated at 0.2, Italics controls previously tested as positive.

Hexavalent Pilot study, protocol number #00-64, ELISA tested against TF(c)-HSA Vaccination 1 2 3 4 5 Ag Patient Ab wkl 2 3 7 12 19 21 31 33 TFc 1 Igm 40 40 40 40 >1280 40 40 40 40 HSA IgG 20 20 >1280 320 80 80 80 160 320 2 IgM NMA 80 160 160 80 40 40 20 20 IgG 10 NMA 10 40 80 NMA NMA 20 40 3 Igm 40 IgG NMA 4 IgM 40 20 320 80 60 80 NMA IgG NMA NMA 640 80 40 160 320 5 IgM NMA 20 160 20 IgG 10 NMA 10 20 6 IgM 40 640 640 640 160 80 80 160 IgG 640  >1280 >1280 >1280 >1280 >1280 >1280 >1280 7 IgM NMA 40 80 640 160 60 40 80 80 IgG 10 320  NMA NMA 40 20 80 20 80 8 IgM NMA NMA 160 160 IgG NMA NMA 10 80 9 IgM NMA NMA NMA 20 NMA NMA NMA NMA IgG NMA NMA 40 NMA 640 80 >1280 160 10 IgM NMA 10 160 160 IgG NMA NMA 40 20 11 IgM 20 IgG NMA 12 Igm 10 40 160 >1280 80 640 80 20 160 IgG NMA NMA NMA NMA >640 20 40 40 80 13 IgM NMA IgG NMA 14 IgM 20 80 40 80 80 40 40 160 160 IgG NMA 40 160 >1280 640 80 80 80 160 15 IgM NMA NMA 20 80 40 40 IgG NMA NMA 10 40 40 40 16 IgM 20 80 >1280 IgG 10 10 80 17 IgM NMA 10 NMA 40 40 40 40 40 160 IgG NMA NMA 40 40 80 40 20 40 18 IgM 40 20 80 640 40 80 80 20 80 IgG NMA NMA NMA 40 160 40 640 40 40 Positive Controls: IgM(19) 1:1280 As from Oct. 19, 2001 end pt. Titers: (+) IgM: 640 IgG (7) 1:640 IgG: 640 (−) Human AB Serum (−) IgM: NMA IgG: NMA

Hexavalent Pilot study, protocol # 00-64 ELISA against glycosylated MUC1-1 (5 sites) Vaccination 1 2 3 4 5 Ag Patient Number Ab wkl 2 3 7 12 19 21 31 MUC 1 Igm NMA 40 >1280 >1280 >1280 >1280 >1280 >1280 1-1G5 IgG NMA NMA 80 160 >1280 640 >1280 >1280 32mer 2 IgM NMA NMA 640 10 NMA 10 20 IgG NMA NMA 10 40 40 20.00 40 160 3 Igm 20 640 IgG NMA 40 4 Igm NMA NMA 320.00 >1280 >1280 >1280 640 640 IgG NMA NMA 10 320 160 >1280 >1280 >1280 5 IgM 10 40 >1280 >1280 IgG NMA NMA 160 >1280 6 IgM NMA 320 >1280 320 320 160 80 IgG NMA 160 >1280 320 160 160 160 7 IgM NMA NMA 40 640 640 80 80 40 IgG NMA NMA 640 >1280 >1280 >1280 >1280 >1280 8 IgM NMA NMA 160 IgG NMA NMA 10 9 IgM NMA NMA 40 160 80 20 20 10 IgG NMA NMA NMA 40 20 10 80 40 10 Igm NMA NMA IgG NMA NMA 11 IgM NMA NMA 10 40 20 160 160 80 IgG NMA NMA 10 320 160 40 40 20 12 Igm 40 40 IgG NMA NMA 13 Igm NMA NMA 20 80 80 320 320 160 IgG NMA NMA 20 20 40 320 320 160 14 Igm 80 20 160 160 40 40 IgG NMA NMA NMA NMA 40 20 15 Igm NMA NMA 640 640 640 10 10 10 IgG NMA NMA 640 640 640 160 80 80 16 Igm NMA 20 IgG NMA 20 17 IgM NMA 20 320 160 40 640 320 80 IgG NMA NMA 10 80 640 40 640 160 Positive Controls: (7) IgM 1: 2560 Controls as of Oct. 15, 2001: (+) Igm: >1280 IgG 1: 2560 Oct. 22, 2001 IgG: >1280 (−) AB Sera (−) IgM: NMA IgG: NMA

Fourth Series of Experiments

Polyvalent Conjugate Vaccine for Cancer

Preliminary Data of Vaccination of High Risk Breast Cancer (BC) Patients (Pts) with a Heptavalent Antigen—Keyhole Limpet Hemocyanin (KLH) Conjugate Plus the Immunologic Adjuvant QS-21.

We have previously shown that following vaccination with single antigen (Ag)-KLH conjugates plus QS-21, the majority of BC pts generate specific antibody (AB) titers. (Clin Ca Res 6:1693, 2000; PNAS 98(6):3270, 2001; Proc ASCO 16:439a, 1997, 18:439a, 1999, 20:271a, 2001) Single Ag's tested have included MUC-1 (various peptide lengths), sTn clustered (c), GloboH and GM2. In an effort to improve and broaden the immune response, we treated BC pts with seven Ag's: 10 mcg each of GM2, GloboH, Lewis^(y); and 3 mcg each of TF(c), sTn(c), Tn(c) and glycosylated MUC-1, (32 amino acid (aa) sequence, glycosylated at 5 sites per 20 aa tandem repeat). Each Ag was conjugated to KLH and mixed with 100 mcg of QS-21. Heptavalent vaccines were administered subcutaneously during weeks 1, 2, 3, 7, and 19. We treated ten patients: median age 48 years (range 43-63 yrs); Stage 1V=3, Stage 2 with ≧4 positive nodes=7. Nine pts have completed immunization. Toxicity was limited to transient grade 2 local skin reactions and grade 1-2 flu-like symptoms. IgM and IgG AB titers were considered positive for each antigen if there was at least an eightfold increase above baseline more than once, during weeks 1-19. Antibody responses are tabulated. (table) MUC1 and TF(c) seem most immunogenic. Flow cytometric analysis (FACS) was obtained pre and post therapy to detect binding of IgM and IgG AB against MCF-7 tumor cells. A positive FACS was defined as at least a threefold increase above baseline and was observed in 6/9 patients for IgM and 0/9 for IgG. Further analyses are ongoing. Our next cohort will evaluate the same antigens conjugated to KLH but with GP-100 as the immunologic adjuvant.

Number of pts with positive AB response/Number of pts evaluable

Ag GM2 Ley MUC1 TF(c) sTn(c) Tn(c) GloboH IgM 2/9 1/9 8/9 8/9 4/9 7/9 6/9 IgG 0/9 1/9 8/9 8/9 1/9 0/9 0/9 Objectives

-   -   Determine immune response against seven antigens and cell lines         expressing these antigens     -   Evaluate toxicity         Background     -   Preclinical data demonstrates that conjugation of an antigen         with keyhole limpet hemocyanin (KLH) and addition of the immune         adjuvant QS-21 augments immunogenicity (Cancer Immunol         Immunother 41:185, 1985; Cancer Res 56:3315, 1996)     -   Following vaccination with single antigen-KLH conjugates plus         QS-21, most breast cancer patients generated IgM and IgG         antibodies against the immunizing antigens (Clin Ca Res 6:1693,         2000; PNAS 98(6):3270, 2001; Proc ASCO 16:439a, 1997, 18:439a.         1999, 20:271a, 2001)     -   These single antigens have included MUC-1 (various peptide         lengths), sTn clustered (c), GloboH and GM2     -   To broaden the immune response, seven antigens were individually         conjugated to KLH and mixed with QS-21 to construct this         heptavalent vaccine         Vaccine Components     -   Antigens         -   Protein: glycosylated MUC-1 (32aa peptide)         -   Gangliosides: GM2, GloboH         -   Carbohydrates: LewisY, sTn(c), Tn(c), TF(c)     -   Immunogenic protein carrier         -   KLH (by the following methods of conjugation):             -   MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester)                 linker for TF(c), sTn(c), Tn(c), and MUC-1             -   MMCCH (4-[4-N-maleimidomethyl] cyclohexane-1-carboxyl                 hydrazide) linker for GloboH and Le^(y)             -   Direct reductive amination for GM2     -   Immunologic Adjuvant         -   QS-21 (purified saponin fraction of tree bark)             Vaccine Components

Doses Antigens* GM2 10 mcg  MUC-1 3 mcg LewisY 10 mcg  GloboH 10 mcg  TF(c) 3 mcg Tn(c) 3 mcg sTn(c) 3 mcg Adjuvant QS-21 100 mcg  *(each conjugated to KLH)

Treatment and Evaluation Plan

WEEK # 1 2 3 5 7 9 13 19 21 (q 3 months) VACCINE 1 2 3 4 5 Blood ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Samples for Immune Response Eligibility Criteria

Breast cancer patients with any one of the following features:

-   -   Stage IV (stable on hormone therapy [tx])     -   Stage IV (no evidence of disease [NED])     -   Stage III     -   Stage II (≧4 positive nodes)     -   Ipsilateral breast or axillary recurrence     -   Rising CA15-3 or CEA levels and NED         Patient Characteristics     -   Total number of patients treated: 10     -   Total number of vaccinations completed: 50         -   (One patient was delayed for unrelated issues)     -   Median age: 48 years (range 43-63 years)     -   Stage

II (with ≧4+ nodes): 7 IV (NED): 2 IV (stable on hormone tx): 1 n = 10 Common Toxicities

-   -   Grade 1-2 injection site skin reactions     -   Grade 1-2-flu-like symptoms     -   No significant laboratory abnormalities     -   No definite autoimmune reactions         Response Criteria: ELISA     -   Serologic Response by ELISA (Enzyme-Linked Immunosorbent Assay)         -   IgM and IgG antibody titers were considered positive for             each antigen if there was a ≧eightfold increase above             baseline more than once, during weeks 1-19     -   Immunologic Response     -   Patient was considered a responder if there was a serologic         response to at least 3 of the 7 antigens         Response Criteria: FACS and CDC     -   Response by FACS (flourescence activated cell sorter) was         considered positive if there was the following increase above         baseline:         -   ≧3-fold increase in percent gated positivity, AND         -   ≧1.5-fold increase on MFI (mean flourescence intensity)     -   Response by CDC (complement-dependent cytotoxicity) was         considered positive if there was a 20% increase above baseline

IMMUNE RESPONSE DATA ELISA GM2 MUC-1 Lewisy GLOBOH TF Tn sTn IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG 1 − − + + − − − − + + − − − − 2 + − + + − − + − + + + − + − 3 + − + + − − + − + + + − + − 4 − − + + + − + + + + + − − − 5 − − − + − − − − − − − − − − 6 − − + + − − + − + + + − + − 7 + − + + − − − − + + + − − − 8 − − + + − − + − + + − − + − 9 − − + + − − + − + + − − − + 10  − − + + − − + − + + − − + − •SUM 3 0 9 10 1 0 7 1 9 9 5 0 5 1

IMMUNE RESPONSE DATA FACS and CDC FACS (IgM) CDC Patient MCF-7 LSC Du-175 MCF-7 LSC 1 + + + − − 2 − + − + + 3 + − − − − 4 + + + + + 5 − − − − − 6 + + − + − 7 + + − − + 8 − + − + − 9 + + + − − 10  − − − + − •SUM 6 7 3 5 3

CONCLUSION

-   -   Vaccination with a heptavalent antigen-KLH conjugate plus QS-21         is well tolerated in breast cancer patients     -   IgM and IgG antibody responses (to at least 3 of 7 antigens)         were observed in 8 patients and 2 patients respectively     -   MUC1 and TF(c) to be appear the most immunogenic of the seven         antigens in this vaccine     -   IgM antibody binding to tumor cells (MCF-7, LSC, Du-145) by FACS         analysis was observed in 6 patients, 7 patients, and 3 patients         respectively     -   There was no consistent evidence of IgG antibody binding to         tumor cells by FACS     -   There was evidence of CDC with the MCF-7 and LSC tumor cell         lines in 5 patients and 3 patients respectively     -   Our next cohort will evaluate the same antigens conjugated to         KLH but with GP-100 as the immunologic adjuvant 

1. A method for inducing antibody production in a subject comprising administering an immunogenic composition comprising an adjuvant and antigens comprising GloboH, GM2, MUC-1, Tn and TF(c), wherein the antigens are individually conjugated to a carrier.
 2. The method of claim 1, wherein MUC-1 is a 32-mer.
 3. The method of claim 2, wherein MUC-1-32-mer is glycosylated.
 4. The method of claim 1, wherein MUC-1 is provided as MUC-1-G5.
 5. The method of claim 1, wherein Tn is provided as Tn(c).
 6. The method of claim 1, wherein Tn is provided as sTn(c).
 7. The method of claim 1, further comprising an antigen selected from the group consisting of MUC-2, MUC-16, Le^(Y), sialyl Le^(a), GM1, GD2, and GD3.
 8. An immunogenic composition comprising an adjuvant and antigens comprising GloboH, GM2, MUC-1, Tn and TF(c), wherein the antigens are individually conjugated to a carrier.
 9. The immunogenic composition of claim 8, wherein MUC-1 is a 32-mer.
 10. The immunogenic composition of claim 9, wherein MUC-1-32-mer is glycosylated.
 11. The immunogenic composition of claim 8, wherein MUC-1 is provided as MUC-1-G5.
 12. The immunogenic composition of claim 8, wherein Tn is provided as Tn(c).
 13. The immunogenic composition of claim 8, wherein Tn is provided as sTn(c).
 14. The immunogenic composition of claim 8, further comprising an antigen selected from the group consisting of MUC-2, MUC-16, Le^(Y), sialyl Le^(a), GM1, GD2, and GD3.
 15. The immunogenic composition of claim 10, wherein MUC-1-32-mer is glycosylated with Tn.
 16. The immunogenic composition of claim 15, wherein said glycosylated MUC-1-32-mer is further conjugated to KLH.
 17. An immunogenic composition comprising an adjuvant and antigens comprising MUC1-Tn conjugate, GloboH and TF(c), wherein said conjugate and each of said GloboH and TF(c) antigens are individually conjugated to a carrier.
 18. The immunogenic composition of claim 17 further comprising GM2 antigen.
 19. The immunogenic composition of claim 17 further comprising the STn antigen.
 20. The immunogenic composition of claim 17, wherein the Muc1-Tn conjugate antigen is conjugated to KLH.
 21. An immunogenic composition comprising an adjuvant and at least two antigens individually conjugated to a carrier, wherein one of the said two antigens is the MUC1-Tn conjugate. 